Dietary essential amino acid supplements increase bone strength by influencing bone mass and bone microarchitecture in ovariectomized adult rats fed an isocaloric low-protein diet.

This study was designed to investigate whether the administration of dietary essential amino acid supplements in adult rats made osteoporotic by estrogen deficiency and reduced protein intake could reverse the deleterious effects caused by these maneuvers. This animal model was selected to mimic the situation observed in elderly women in whom estrogen deficiency and/or low-protein intake (but also calcium and vitamin D deficiency) are known to contribute to the pathogenesis of osteoporosis. Six-month-old rats were ovariectomized (OVX) and fed an isocaloric 2.5% casein diet for 10 weeks or sham-operated (SHAM) and fed an isocaloric 15% casein diet. The animals fed the 2.5% casein diet were given isocaloric supplements of essential amino acids in similar relative proportion to that of casein at doses of 2.5% or 5% of total diet for an additional 16 weeks. Vertebrae, femur, and tibia bone mineral density (BMD); ultimate strength; and microtomographic histomorphometry were evaluated before and after dietary essential amino acid supplements. Essential amino acid supplements increased vertebrae, femur, and tibia bone strength in OVX rats fed a low-protein diet. The mechanical changes induced by this dietary isocaloric supplement were associated with the prevention of a further BMD decrease or even with some increases and changes in microarchitecture such as from a rod to a plate trabecular spacial configuration and increased cortical thickness. Higher insulin-like growth factor (IGF) I levels, as well as greater bone formation and reduced bone resorption as assessed by biochemical markers of bone remodeling, were found in rats receiving essential amino acid supplements. In conclusion, dietary essential amino acid supplements increased bone strength through modifications of BMD, trabecular architecture, and cortical thickness possibly by an IGF-I-mediated process.     

Protein undernutrition-induced bone loss is associated with decreased IGF-I levels and estrogen deficiency.

Protein undernutrition is a known factor in the pathogenesis of osteoporotic fracture in the elderly, but the mechanisms of bone loss resulting from this deficiency are still poorly understood. We investigated the effects of four isocaloric diets with varying levels of protein content (15, 7.5, 5, and 2.5% casein) on areal bone mineral density (BMD), bone ultimate strength, histomorphometry, biochemical markers of bone remodeling, plasma IGF-I, and sex hormone status in adult female rats. After 16 weeks on a 2.5% casein diet, BMD was significantly decreased at skeletal sites containing trabecular or cortical bone. Plasma IGF-I was decreased by 29-34% and no estrus sign in vaginal smear was observed. To investigate the roles of estrogen deficiency and protein undernutrition, the same protocol was used in ovariectomized (OVX) or sham-operated (SHAM) rats, pair-fed isocaloric diets containing either 15 or 2.5% casein. Trabecular BMD was decreased by either manipulation, with effects appearing to be additive. Cortical BMD was decreased only in rats on a low-protein diet. This was accompanied by an increased urinary deoxypyridinoline excretion without any change in osteocalcin levels, suggesting an uncoupling between resorption and formation. Isocaloric protein undernutrition decreased bone mineral mass and strength. This effect might be related to decreased plasma IGF-I and/or estrogen deficiency with a consequent imbalance in bone remodeling.     

Defective implant osseointegration under protein undernutrition: prevention by PTH or pamidronate.

Protein deficiency is associated with impaired titanium osseointegration. We studied whether systemic treatment with PTH or pamidronate could influence the resistance to pull-out of titanium rods implanted into rats proximal tibia under normal and isocaloric low protein intake. PTH or pamidronate prevented the deleterious effects of protein undernutrition on bone microarchitecture close to the implant and on mechanical fixation. PTH even significantly improved implant osseointegration. INTRODUCTION: Protein deficiency is highly prevalent among elderly patients hospitalized in orthopedic wards. Reduced protein intake impairs titanium osseointegration in rats. Whether stimulator of bone formation or inhibitor of bone resorption could improve implant osseointegration under protein deprivation is not known. We studied the effects of systemic treatment with PTH or pamidronate on the resistance to pull-out of titanium rods implanted into rats proximal tibia under normal and isocaloric low protein intake. MATERIALS AND METHODS: We measured the resistance to pull-out 1-mm-diameter titanium rods implanted into the proximal tibias of 49 adult female rats receiving a normal or an isocaloric low protein diet. After 2 wk on either diet, the implants were inserted, and the rats received PTH(1-34), pamidronate or saline vehicle for 8 wk. The tibias were removed for microCT morphometry, followed by the evaluation of pull-out strength. RESULTS: Pull-out strength was lower in rats fed an isocaloric low protein diet compared with rats fed a normal protein intake (-29%). PTH and pamidronate significantly increased pull-out strength in animals fed a normal or a low protein diet, the effect of PTH being of higher magnitude. The PTH- or pamidronate-mediated increase in pull-out strength was associated with significant increases of relative bone volume, bone-to-implant contact, and trabecular thickness, whereas trabecular spacing was reduced, in the vicinity of the implants. CONCLUSIONS: We confirmed that isocaloric low protein intake impairs titanium implant osseointegration. PTH or pamidronate prevented the deleterious effects of protein undernutrition and even significantly improved the implant osseointegration. These results indicate that systemic administration of PTH or pamidronate could be considered for preventing uncemented arthroplasty loosening in protein undernourished patients.     

Bone density and shape as determinants of bone strength in IGF-I and/or pamidronate-treated ovariectomized rats

Areal bone mineral density (BMD) is a major determinant of bone strength and thereby of fracture risk. Other factors including trabecular microarchitecture and bone dimensions also contribute to bone strength. To investigate the relative importance for bone strength of BMD and bone dimensions, the relations between strength and the latter variables were evaluated under different experimental conditions in ovariectomized rats. Bone strength was assessed in compression and bending with measurement of BMD by dual-energy X-ray absorptiometry. Interventions were designed to increase trabecular BMD in rats with estrogen deficiency-induced bone loss (OVX) by treatment with pamidronate, an inhibitor of bone resorption, or to modify bone dimensions, particularly diameter, by administration of the growth factor IGF-I. In OVX rats, pamidronate treatment increased BMD with a commensurate increase in bone strength at the level of lumbar vertebrae and femoral neck (r=0.789,p<0.0001 andr=0.535,p<0.001, respectively). IGF-I increased the external diameter of midshaft tibia and femoral neck, which also correlated with bone strength (r=0.678,p<0.0001 andr=0.507,p<0.0002, respectively). Thus, both bone dimensions and BMD contributed to the determination of bone strength. In conclusion, adult rats with estrogen deficiency-induced bone loss represent a useful experimental model for investigating bone strength and its determinants such as BMD and external bone dimensions.     

Dietary protein deficiency induces osteoporosis in aged male rats.

Low dietary intake is common in elderly males with low femoral neck areal bone mineral density (BMD). To evaluate the selective influence of a low-protein diet in the pathogenesis of osteoporosis in males and to uncover early and late adaptation of bone cells to protein deficiency, 8-month-old male rats were pair-fed a control (15% casein) or isocaloric low-protein (2.5% casein) diet for 1 or 7 months. BMD, bone ultimate strength, stiffness, and absorbed energy were measured in tibia proximal metaphysis and diaphysis. After double-labeling, histomorphometric analysis was performed at the same sites. Serum osteocalcin, insulin-like growth factor I (IGF-I), and urinary deoxypyridinoline excretion were measured. In proximal tibia, isocaloric low-protein diet significantly decreases BMD (12%), cancellous bone mass (71%), and trabecular thickness (Tb.Th; 30%), resulting in a significant reduction in ultimate strength (27%). In cortical middiaphysis, a low-protein diet decreases BMD (9%) and enlarges the medullary cavity (36%), leading to cortical thinning and lower mechanical strength (20%). In cancellous bone, protein deficiency transiently depresses the bone formation rate (BFR; 60%), osteoid seam thickness (15%), and mineral apposition rate (MAR; 20%), indicating a decrease in osteoblast recruitment and activity. Cortical loss (15%) results from an imbalance between endosteal modeling drifts with impaired BFR (70%). From the first week of protein deficiency, osteocalcin and IGF-I levels drop significantly. Bone resorption activity and urinary deoxypyridinoline remain unchanged throughout the experiment. Protein deficiency in aged male rats induces cortical and trabecular thinning, and decreases bone strength, in association with a remodeling imbalance with a bone formation impairment and a decrease in IGF-I levels.     

Different responsiveness of alveolar and tibial bone to bone loss stimuli.

Mandibular and systemic bone loss are poorly associated. We compared the effect of isocaloric protein undernutrition and/or ovariectomy on BMD and microstructure of mandibular alveolar and proximal tibia sites in adult rats. Mandibular bone was significantly less affected. INTRODUCTION: Whether mandibular bone and axial or peripheral skeleton respond similarly to systemic bone loss remains a subject of controversy. We have previously shown that mechanical loading during mastication influences bone mass and architecture of the mandibular alveolar bone. Isocaloric protein undernutrition and ovariectomy are known to cause bone loss and deterioration of bone microarchitecture at various axial and peripheral skeletal sites. We studied how the mandible, which is subjected to heavy, abrupt, and intermittent forces during mastication, responds to low-protein intake and/or ovariectomy and compared this response to that of the proximal tibia in adult rats. MATERIALS AND METHODS: Forty-four 6-month-old female Sprague-Dawley rats underwent transabdominal ovariectomy (OVX; n=22) or sham operation (n=22) and were pair-fed isocaloric diets containing either 15% or 2.5% casein (sham 15%, n=11; sham 2.5%, n=11; OVX 15%, n=11; and OVX 2.5%, n=11) for 16 weeks. BMD and bone microarchitecture parameters (e.g., bone volume fraction [BV/TV] and trabecular thickness and number) of the mandible and the proximal tibia were measured at the end of the experiment using DXA and microCT. RESULTS: Mandibular alveolar bone was negatively influenced by both protein undernutrition and OVX, but to a significantly lesser extent than the proximal tibia. In sham-operated animals, low-protein intake led to a 17.3% reduction of BV/TV in the mandible and 84.6% in the tibia (p<0.001). In normal protein diet-fed animals, OVX led to a reduction of BV/TV of 4.9% in the mandible but 82% in the tibia (p<0.001). In the mandible, protein undernutrition resulted in thinner trabeculae (p<0.05), whereas OVX led to a reduction of trabecular number (p<0.05). CONCLUSIONS: Mandibular alveolar bone was found to be less sensitive to either protein undernutrition or OVX than the proximal tibia spongiosa. We hypothesize that the mechanical loading of the alveolar process during mastication may protect the alveolar bone from the detrimental effects observed in other skeletal sites, such as the proximal tibia. Morphological and embryological differences between the two skeletal sites might also play a role.     

Low protein intake is associated with impaired titanium implant osseointegration.

Low protein intake is highly prevalent among orthopaedic elderly patients. We studied the effects of an isocaloric low protein diet on the resistance to pull-out of titanium rods implanted into rats proximal tibia. Isocaloric low protein intake impairs titanium implant osseointegration, with a decreased strength needed to completely loose the implant and altered bone microarchitecture in its vicinity. INTRODUCTION: Low protein intake is highly prevalent among elderly patients in orthopaedic wards and could retard fracture healing. It was previously shown that reduced protein intake decreases bone strength. Whether dietary protein intake could influence titanium implant osseointegration is unknown. We studied the effects of an isocaloric low protein diet on the resistance to pull-out of titanium rods implanted into rats proximal tibia. MATERIALS AND METHODS: Forty-eight 11-month-old female rats were fed isocaloric diets containing 2.5% (low protein) or 15% (normal protein) casein from 2 weeks before the implantation of a 1-mm-diameter cylindrical titanium rod in the proximal metaphysis of each tibia. Four, 6, and 8 weeks after implantation, the tibias were removed for microCT histomorphometry to quantify bone-to-implant contact and bone trabecular microarchitecture around the implant. Resistance to implant pull-out was tested by recording the maximal force necessary to completely loosen the implant. RESULTS: Pull-out strength was significantly lower in rats fed an isocaloric low protein diet by 6 and 8 weeks after implantation (-43%, p < 0.001 and -42%, p < 0.001, respectively) compared with rats fed a normal protein diet. Bone-to-implant contact was significantly lower in the low protein group 8 weeks after implantation (p < 0.05). Bone-to-implant contact and pull-out strength were correlated (r2= 0.57, p < 0.0001). BV/TV around the implant was 19.9 +/- 2.2% (SE) versus 31.8 +/- 3.3% (p < 0.05) at 6 weeks and 20.1 +/- 1.9% versus 29.8 +/- 3.2% (p < 0.05) at 8 weeks after implantation in the low protein and normal protein intake groups, respectively. Trabecular thickness was 96.2 +/- 3.7 versus 113.0 +/- 3.6 microm (p < 0.01) at 6 weeks and 101.4 +/- 2.7 versus 116.2 +/- 3.3 microm (p < 0.01) at 8 weeks in the corresponding groups. In a structure model index analysis, there was a significant shift to a more rod-like pattern in the low protein diet groups. All these changes were associated with lower plasma IGF-I levels. CONCLUSIONS: Isocaloric low protein intake impairs titanium implant osseointegration, with decreased strength needed to completely loosen the implant and altered bone microarchitecture in the vicinity of the implant.     

Recovery of proximal tibia bone mineral density and strength, but not cancellous bone architecture, after long-term bisphosphonate or selective estrogen receptor modulator therapy in aged rats.

Various bisphosphonates and the selective estrogen receptor modulator (SERM) raloxifene are approved treatments of postmenopausal osteoporosis. They increase bone mineral density (BMD), decrease bone turnover, and reduce vertebral fracture incidence through different cellular mechanisms. We investigated the bone cellular activities, architecture, mineral content/density, and strength of ovariectomized (ovx) rats on a long-term bisphosphonate or SERM treatment, at doses of either agent correcting bone strength. Eleven weeks postovariectomy, 6-month-old rats were treated with the SERM MDL 103,323 or with the bisphosphonate pamidronate for 5 months. Doses of pamidronate and MDL 103,323 were selected from previous studies showing correction of the ovx-induced decrease of ultimate strength of proximal tibia. Ultimate and yield strengths, BMD, and histomorphometric parameters were all quantified at the same site, i.e., the proximal tibia metaphysis. Long-term pamidronate decreases bone turnover and bone formation activity, leading to trabecular thinning. MDL 103,323 decreases bone turnover to a lesser extent, and slightly protects trabecular architecture by uncoupling bone resorption and formation activities. The yield strength is corrected by pamidronate, but not by MDL 103,323 treatment. However, neither compound restores the ovariectomy-induced cancellous bone loss. Total tissue area and cortical thickness are unchanged with pamidronate or MDL 103,323 treatment, indicating that cortical bone mass, thickness, and cross-sectional area are not modified. The discrepancy between proximal tibia BMD and mechanical resistance to fracture modifications, on the one hand, and cancellous bone volume, on the other hand, could be due to changes in the degree of mineralization of bone matrix and/or of the intrinsic properties of the bone matrix.     

Intrinsic bone tissue properties in adult rat vertebrae: modulation by dietary protein

Bone strength depends on bone mass, geometry, microarchitecture, and intrinsic tissue quality. Whether intrinsic bone tissue properties could be influenced by changes in dietary protein is not known. To address this issue, nanoindentation tests were performed on the lateral, anterior, and posterior site of L5 vertebral bodies in adult female rats fed a normal protein containing diet and in ovariectomized (OVX) rats receiving an isocaloric low protein diet with or without isocaloric essential amino acids supplements. The tissue properties varied significantly between the different sites (anterior, posterior, lateral), suggesting possible effects of heterogeneous stress distribution on the vertebrae in vivo. Isocaloric low protein intake associated with ovariectomy led to significant decreases of indentation modulus, hardness, and dissipated energy on the posterior vertex. Axial compression tests of adjacent vertebral bodies were correlated with the indentation results. Correlations between macroscopic mechanical data obtained by axial compression of vertebral body, and intrinsic tissue properties measured by nanoindentation test suggest that postelastic behavior strongly varied with material fragility detected on the tissue level. Macroscopic stiffness however may be dominated by bone geometry changes and less by variations of intrinsic bone tissue properties. Combining parameters of tissue properties and bone mineral density was highly predictive of vertebral body ultimate strength. Besides geometry and microarchitecture, intrinsic bone tissue properties are important determinants of the mechanical competence of rat vertebrae. Changes in intrinsic tissue properties could thus contribute to the increased bone fragility found in protein undernutrition.     

Alendronate administration and skeletal response during chronic alcohol intake in the adolescent male rat.

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for alpha-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 +/- 13 mg/dl (E) and 203 +/- 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.     

Treatment of experimental renal osteodystrophy with pamidronate

We evaluated the effects of the bisphosphonate pamidronate on bone histomorphometry, structure and strength in male rats with uninephrectomy or with chronic renal disease induced by 5/6 nephrectomy. In rats with chronic renal disease the plasma urea, phosphate and parathyroid hormone levels were significantly increased compared to rats with a uninephroctomy and none of these parameters was affected by pamidronate treatment. In the femoral midshaft, chronic renal disease reduced cortical bone mineral density and content. No difference was observed in the breaking load of the femoral midshaft. In the distal femur, a high-turnover renal osteodystrophy was found but pamidronate suppressed this bone turnover and increased bone mineral content. Treatment had no effect on chronic disease-induced augmentation of osteoid volume or fibroblast surface. These studies show that in this model of stage 3 renal disease, pamidronate increased mineral content in the femoral midshaft and distal metaphysis primarily by adding bone to endocortical and trabecular surfaces but did not reduce osteitis fibrosa.     

Comparison of dried plum supplementation and intermittent PTH in restoring bone in osteopenic orchidectomized rats

Summary Bone loss was confirmed after 90 days in 50 6-month-old male Sprague Dawley rats that were sham-operated or orchidectomized (ORX). In this study, we have shown that dried plum (DP) has potent effects on bone in terms of bone mass, microarchitecture, and strength in osteopenic male rats. Although these changes may be mediated through the suppression of bone resorption, the fact that the restoration in some of the bone structural and biomechanical parameter shares some similarities with parathyroid hormone (PTH) should not be overlooked. Further investigation is needed on a mechanistic level to clarify the influence of DP on bone metabolism. Introduction This study was designed to investigate the extent to which DP reverses bone loss in osteopenic ORX rats and to compare its effects to PTH. Materials and methods Fifty, 6-month-old male Sprague Dawley rats were sham-operated or ORX, and bone loss was confirmed after 90 days. The ORX groups were assigned to control (AIN-93M) diet, 25% DP diet, or PTH (80 μg/kg) for 90 days. Results DP induced an 11% increase in vertebral and femoral BMD compared to ORX-controls. BMD in the PTH-treated group was increased by 20.7% (vertebra) and 17.9% (femur). Vertebral trabecular bone volume (BV/TV) and number were increased by DP and trabecular separation was decreased compared to controls, which were similar to PTH. Alterations in trabecular bone of the femur were similar to those in the vertebra, but DP did not restore BV/TV to the same extent. Cortical thickness was improved by DP and further enhanced by PTH. DP tended to decrease urinary deoxypyridinoline and calcium, but did not alter alkaline phosphatase or osteocalcin. Conclusion We conclude that though the degree of improvement was not equivalent to PTH with regard to all parameters, DP reverses bone loss due to ORX and the mechanisms should be further investigated.     

Effects of osteoporosis medications on bone quality

In clinical practice, the quantitative evaluation of bone tissue relies on dual-energy X-ray absorptiometry (DXA) measurements of bone mineral density (BMD) values, which are closely associated with the risk of osteoporotic fracture. However, only a small fraction of the antifracture effect of bone resorption inhibitors is ascribable to BMD gains (4% with raloxifene and 16-28% with alendronate and risedronate). Bone quality encompasses a number of bone tissue properties that govern mechanical resistance, such as bone geometry, cortical properties, trabecular microarchitecture, bone tissue mineralization, quality of collagen and bone apatite crystal, and presence of microcracks. All these properties are dependent on bone turnover and its variations. In populations, the decreases in bone resorption markers achieved with resorption inhibitors may predict in part the decrease in fracture risk. At the spine, however, this correlation exists down to a 40% fall in bone resorption markers; larger drops did not provide further protection against fractures in patients taking risedronate in one evaluation of this relationship. Osteoporosis medications can exert favorable effects on bone size and cortical thickness. Such effects have been documented with teriparatide (PTH 1-34), which is the unique purely anabolic treatment for osteoporosis available to date. More surprising are the favorable effects on bone size seen with some of the bone resorption inhibitors such as neridronate in adults with osteogenesis imperfecta. Similarly, estrogens and alendronate can increase femoral neck size in postmenopausal women. Preservation of the trabecular microarchitecture was demonstrated first with risedronate and subsequently with alendronate. In placebo-controlled studies, a deterioration in trabecular microarchitecture occurred within 1 to 3 years in the placebo groups but not in the bisphosphonate groups. Teriparatide, in contrast, improves trabecular microarchitecture, in particular by increasing connectivity and improving the plate-rod distribution. The minerals within trabecular or cortical bone can be evaluated using microradiography or synchrotron micro-computed tomography. Marked or prolonged secondary mineralization may result in poor bone quality. Increased bone mineralization is among the key effects of bone resorption inhibitors, most notably bisphosphonates. Prolonged use of the most potent bisphosphonates may lead to unwanted effects related to excessive mineralization. Microcracks may play a physiological role; however, a large number of microcracks may be deleterious via an effect on osteocytes. Excessive mineralization may promote the development of multiple microcracks. Studies of bone crystal and collagen properties with several bone resorption inhibitors, including risedronate and raloxifene, showed no harmful effects. An increasing number (several hundreds) of mandibular osteonecrosis associated with bisphosphonate therapy has been reported. The typical patient was receiving injectable bisphosphonate therapy for bone cancer and had undergone dental work shortly before bisphosphonate administration. The mechanism of this adverse effect is poorly understood.     

Subregion analysis of the rat femur: A sensitive indicator of changes in bone density following treatment with thyroid hormone or bisphosphonates

Measurement of bone mineral density (BMD) by dual X-ray absorptiometry (DXA) is a precise and accurate way to assess changes in BMD due to a variety of causes. However, the degree of bone loss may vary depending on the skeletal site examined. We postulated that interventions that change bone density would have a different effect on an area rich in trabecular bone, such as the distal femur, than on other subregions of the femur. Male Sprague-Dawley rats (325–350 g) were treated with triiodothyronine (T₃), a bisphosphonate (pamidronate), or placebo for 21 days and then sacrificed. Ex vivo BMD of the proximal, distal, mid and total femur were measured by DXA. We found that mean BMD of hyperthyroid rats was significantly lower than controls at all femoral subregions. However, the difference in mean BMD between hyperthyroid and control rats was greatest at the distal femur (8.6%). In rats treated with bisphosphonate, mean BMD was significantly higher than controls at the proximal, distal, and total femur. The difference in mean BMD between controls and rats treated with bisphosphonate was greatest at the distal femur (31.8%). Furthermore, pamidronate (APD)-treated rats had lower mean mid-femur BMD than controls. We conclude that changes in BMD after treatment with bisphosphonate or T₃ are greatest at the distal femur subregion, and that treatment with bisphosphonate may cause a slight reduction in midfemur BMD. Future studies examining changes in BMD in the rat femur after interventions that alter mineral metabolism should include subregion analysis.Portions of the data presented here were presented at the Fourteenth Annual Meeting of the American Society for Bone and Mineral Research, Minneapolis, Minnesota, September 30–October 4, 1992.     

Minodronic acid (ONO-5920/YM529) prevents decrease in bone mineral density and bone strength, and improves bone microarchitecture in ovariectomized cynomolgus monkeys

This study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone microarchitecture and bone strength in ovariectomized (OVX) cynomolgus monkeys. Skeletally mature female cynomolgus monkeys, aged 9-17 years, were ovariectomized or sham-operated. Minodronic acid was administered orally once a day in doses of 0, 0.015, and 0.15 mg/kg from the day after surgery for 17 months. Bone resorption markers (urinary N-terminal cross-linking telopeptide of type I collagen and deoxypyridinoline), bone formation markers (serum osteocalcin and bone alkaline phosphatase) and lumbar vertebral BMD were measured at baseline and at 4, 8, 12 and 16 months after surgery. Treatment with minodronic acid dose-dependently inhibited OVX-induced increase in bone turnover markers and decrease in lumbar vertebral BMD, and minodronic acid at 0.15 mg/kg completely prevented these changes. At 17 months after surgery, minodronic acid also suppressed bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (OS/BS, MS/BS, MAR, BFR/BS, and BFR/BV) in the lumbar vertebral bodies and tibia. In the mechanical tests, ultimate load on lumbar vertebral bodies and femoral neck of the OVX-control animals were significantly reduced compared to the sham animals. Minodronic acid prevented these reductions in bone strength at 0.15 mg/kg. There was significant correlation between BMD and bone strength, suggesting that the increase in bone strength was associated with the increase in BMD produced by minodronic acid. In micro-CT analysis of the lumbar vertebral bodies, minodronic acid improved trabecular architecture, converting rod structures into plate structures, and preventing the increase in trabecular disconnectivity at 0.15 mg/kg. In conclusion, similar to patients with postmenopausal osteoporosis, reduction in bone strength of lumbar vertebral bodies and femoral neck was clearly demonstrated in OVX cynomolgus monkeys. Minodronic acid prevented these reductions at a once-daily oral administration. Also, minodronic acid prevented OVX-induced changes in bone turnover, bone mass and bone microarchitecture. Long-term minodronic acid treatment was well tolerated and no adverse effects could be detected. These results suggest that minodronic acid may be a clinically useful drug for osteoporosis.     

Intravenous pamidronate prevents femoral bone loss and renal stone formation during 90-day bed rest.

Long-term bed rest has potential risks of bone loss and renal stone formation. We examined the effects of resistive exercise and intravenous pamidronate on BMD, bone turnover, urinary calcium, and renal stone formation in 25 healthy males during 90-day bed rest. Pamidronate prevented femoral bone loss and renal stone formation, but resistive exercise showed little effects. INTRODUCTION: Long-term bed rest increases the risks of bone loss and urinary stone formation. Resistive exercise increases bone formation, and bisphosphonates reduce bone resorption. However, the effects of muscle exercise and bisphosphonates have not been examined side-by-side. The objectives of this study are to compare the effects of pamidronate with resistive exercise on BMD and renal stone formation during prolonged bed rest. MATERIALS AND METHODS: Twenty-five male white volunteers, 26-45 years of age, were randomly assigned to the control (n = 9), exercise (n = 9), and pamidronate (n = 7) groups and underwent 90-day 6 degrees head-down tilt bed rest. Exercise group performed squats and heel raises on a flywheel device for 30 minutes every 3 days. Pamidronate (60 mg) was administered intravenously 14 days before bed rest. BMD of the head, forearm, lumbar spine, and proximal femur; biochemical bone markers; calcium (Ca) metabolism; and abdominal radiographs were examined during 90 days of bed rest and 360 days of reloading. RESULTS: In controls, proximal femoral BMD decreased, and bone resorption markers and urinary Ca increased during bed rest, along with development of renal stones in two of nine subjects. Resistive exercise increased bone formation but was unable to prevent femoral BMD decrease and increases in bone resorption and urinary Ca during bed rest, with formation of renal stones in four of nine subjects. Pamidronate maintained femoral BMD, reduced bone resorption and urinary Ca, and completely prevented renal stone formation. CONCLUSIONS: Resistive exercise increased bone formation but could not reduce bone resorption and the risk of renal stones. In contrast, inhibition of bone resorption by pamidronate could preserve bone mineral and reduce the risk of renal stone formation during prolonged bed rest.     

Consumption of different sources of omega-3 polyunsaturated fatty acids by growing female rats affects long bone mass and microarchitecture

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) consumption has been reported to improve bone health. However, sources of ω-3 PUFAs differ in the type of fatty acids and structural form. The study objective was to determine the effect of various ω-3 PUFAs sources on bone during growth. Young (age 28d) female Sprague-Dawley rats were randomly assigned (n=10/group) to a high fat 12% (wt) diet consisting of either corn oil (CO) or ω-3 PUFA rich, flaxseed (FO), krill (KO), menhaden (MO), salmon (SO) or tuna (TO) for 8 weeks. Bone mass was assessed by dual-energy X-ray absorptiometry (DXA) and bone microarchitecture by micro-computed tomography (μCT). Bone turnover markers were measured by enzyme immunoassay. Lipid peroxidation was measured by calorimetric assays. Results showed that rats fed TO, rich in docosahexaenoic acid (DHA, 22:6ω-3) had higher (P<0.009) tibial bone mineral density (BMD) and bone mineral content (BMC) and lower (P=0.05) lipid peroxidation compared to the CO-fed rats. Reduced lipid peroxidation was associated with increased tibial BMD (r2=0.08, P=0.02) and BMC (r2=0.71, P=0.01). On the other hand, rats fed FO or MO, rich in alpha-linolenic acid (ALA, 18:3ω-3), improved bone microarchitecture compared to rats fed CO or SO. Serum osteocalcin was higher (P=0.03) in rats fed FO compared to rats fed SO. Serum osteocalcin was associated with improved trabecular bone microarchitecture. The animal study results suggest consuming a variety of ω-3 PUFA sources to promote bone health during the growth stage.     

High dietary phosphate intake reduces bone strength in the growing rat skeleton.

Nutrition influences peak bone mass development in early adulthood. The effect of high dietary phosphate intake on the growing skeleton of 1-month-old male rats (n = 30) was assessed in an 8-week intervention. High dietary phosphate intake increased bone remodeling and impaired bone material properties, diminishing bone mechanical strength. INTRODUCTION: High dietary phosphate intake is typical in the Western diet. Abundant phosphate intake enhances parathyroid secretion and bone metabolism. To study the influence of high dietary phosphate intake on growing bone homeostasis and structure, we submitted growing rats to experimental diets that varied in their phosphate content. MATERIALS AND METHODS: One-month-old intact male rats (n = 30) were fed a control diet (Ca:P 1:1) or an experimental diet of either Ca:P 1:2 or Ca:P 1:3 for 8 weeks. At the beginning and the end of the study period, the right femurs were measured using DXA. Double labeling with tetracycline injection was performed 12 and 2 days before death. After death, hind legs were cut loose. Left femurs were processed for histomorphometry. Right femurs were measured with pQCT. Mechanical testing was performed on the right femoral neck and tibial shaft. Six right tibias were analyzed with microCT. Serum PTH, calcium, and phosphate contents were analyzed. RESULTS: High-phosphate intake impaired growth of the animal, limited bone longitudinal growth, and restricted femur BMC and BMD build-up. Osteoclast number, osteoblast perimeter, and mineral apposition rate were increased, and trabecular area and width were decreased. Phosphate decreased femur midshaft total bone BMD, cortical bone BMD, and mean cortical thickness. High-phosphate diet reduced femoral neck and tibial shaft ultimate strength and tibia stiffness and toughness. In addition, serum PTH increased. CONCLUSIONS: High dietary phosphate intake reduced growth, skeletal material, and structural properties and decreased bone strength in growing male rats. Adequate calcium could not overcome this.     

Do bisphosphonates reduce early micromotion and Periprosthetic bone loss in total knee arthroplasty? A review of the evidence

Early micromotion of implant components and periprosthetic bone loss in patients undergoing total knee arthroplasty are thought to contribute to late aseptic loosening. In the pursuit of longer implant survival, the administration of bisphosphonates may be advocated as a means to buffer implants against microinstability and periprosthetic bone loss. A bibliographic search identified one metaanalysis and two randomised controlled trials dealing with this topic. Current evidence supports the hypothesis that the inhibiting effects of bisphosphonates on bone resorption reduce implant micromotion and periprosthetic bone loss at the one-year follow-up. Tested bisphosphonates include clodronate, pamidronate and alendronate. However, a decline in periprosthetic BMD is observed at the three-year follow-up following a sixmonth course of bisphosphonate administration. Length of follow-up in available studies is currently too short to determine whether bisphosphonates increase the longevity of implants. Furthermore, the optimal dose, modality and length of bisphosphonate administration have yet to be determined.     

Long-term minodronic acid (ONO-5920/YM529) treatment suppresses increased bone turnover, plus prevents reduction in bone mass and bone strength in ovariectomized rats with established osteopenia

The present study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone histomorphometry and bone strength in ovariectomized (OVX) rats. Female F344/DuCrj rats, aged 14 weeks, were OVX or sham operated. After 3 months, the OVX rats showed an increase in bone turnover, and a decrease in bone mass and bone strength. Minodronic acid was administered orally once a day for 12 months at doses of 0, 0.006, 0.03 and 0.15 mg/kg from 3 months after OVX. Minodronic acid dose-dependently inhibited the decrease in BMD of lumbar vertebrae and femur. In the femur, treatment with 0.15 mg/kg minodronic acid increased the BMD of distal and mid sites to sham levels. Minodronic acid dose-dependently suppressed OVX-induced increase in urinary deoxypyridinoline, a bone resorption marker, after a month of treatment and these effects were maintained for 12 months of treatment. Minodronic acid also decreased serum osteocalcin, a bone formation marker. In bone histomorphometric analysis after 12 months of treatment, OVX rats showed an increase in bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (MS/BS and BFR/BV) at lumbar vertebral bodies. Minodronic acid suppressed the OVX-induced increase in bone turnover at tissue level. Trabecular bone volume, trabecular thickness and trabecular number of lumbar vertebral bodies were decreased after OVX. Minodronic acid increased these structural indices, indicating that it prevented the deterioration in trabecular architecture. In a mechanical test at 12 months of treatment, ultimate load of lumbar vertebral bodies and mid femur in the OVX-control group was decreased compared to the sham group. Minodronic acid prevented the reduction in bone strength at both sites. In particular, in the mid femur, treatment with 0.03 and 0.15 mg/kg minodronic acid increased bone strength to sham levels or greater. In conclusion, minodronic acid suppressed increased bone turnover, plus prevented the decrease in BMD, deterioration of bone microarchitecture and reduction in bone strength in OVX rats with established osteopenia. These results suggest that minodronic acid may be clinically useful for treatment of osteoporosis.