Effects of dried plum supplementation on bone metabolism in adult C57BL/6 male mice.

Dietary supplementation of dried plum (DP) prevents bone loss and restores bone mass in osteopenic animal models. This study was designed to determine the effects of DP supplementation on bone metabolic activity over time using adult (6-month-old) male C57BL/6 mice (n = 40) receiving control (CON = AIN93 M) or CON+DP 25 % (w/w) diets for 4 or 12 weeks. After 4 weeks of treatment, animals consuming the DP diet had a higher whole-body bone mineral density, vertebral trabecular bone volume (BV/TV), and femoral cortical thickness compared to the CON animals. In the distal metaphysis of the femur, BV/TV was increased in the DP-treated animals, but only after 12 weeks. Bone histomorphometric analyses revealed that DP decreased osteoblast surface (67 %) and osteoclast surface (62 %) at 4 weeks, but these surfaces normalized to the CON animals by 12 weeks. Coincident with these changes, the mineralizing surface (MS/BS) and cancellous bone formation rate (BFR/BS) were reduced at 4 weeks in the DP group compared to the CON, but by 12 weeks of DP supplementation, BFR/BS (~twofold) and MS/BS (~1.7-fold) tended to be increased (p < 0.10). The relative abundance of RNA for key regulators of osteoblast and osteoclast differentiation and indicators of osteoblast activity were reduced in the DP group at 4 weeks with no difference between groups at 12 weeks. These results indicate that supplementing the diet with DP initially suppressed cancellous bone turnover, but a biphasic response occurs over time, resulting in a positive effect on bone mass and structure.

Enhanced alveolar bone loss in a model of non-invasive periodontitis in rice rats.

OBJECTIVE: The rice rat (Oryzomys palustris) develops periodontitis-like lesions when fed a diet rich in sucrose and casein (H-SC). We aimed to establish whether this model can accurately mimic the development of human periodontitis. MATERIALS AND METHODS: For this purpose, 28-day-old rice rats (15/group) were assigned to standard (STD) or H-SC diets and sacrificed after 6, 12, and 18 weeks. Jaws were processed for morphometric, histometric, histologic, histomorphometric, and micro-CT analyses. RESULTS: We found a progressive increase in horizontal alveolar bone loss (ABL) with age in maxillae of rats fed the STD diet as determined by morphometry. The H-SC diet exacerbated horizontal ABL at the palatal surface at 12 and 18 weeks. Furthermore, increased vertical ABL was detected in mandibles and maxillae of rats fed the H-SC diet for 12 and/or 18 weeks by histometry and micro-CT. Remarkably, the H-SC diet significantly increased bone remodeling at the interproximal alveolar bone of mandibles from rats fed for 6 weeks, but not in those fed for longer periods. CONCLUSIONS: These findings indicate that the H-SC diet induced a transient increase in alveolar bone remodeling, which is followed by ABL characteristic of moderate periodontitis.

Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.

The FAM20 family of secreted proteins consists of three members (FAM20A, FAM20B, and FAM20C) recently linked to developmental disorders suggesting roles for FAM20 proteins in modulating biomineralization processes. The authors report here findings in knockout mice having null mutations affecting each of the three FAM20 proteins. Both Fam20a and Fam20c null mice survived to adulthood and showed biomineralization defects. Fam20b (-/-) embryos showed severe stunting and increased mortality at E13.5, although early lethality precluded detailed investigations. Physiologic calcification or biomineralization of extracellular matrices is a normal process in the development and functioning of various tissues (eg, bones and teeth). The lesions that developed in teeth, bones, or blood vessels after functional deletion of either Fam20a or Fam20c support a significant role for their encoded proteins in modulating biomineralization processes. Severe amelogenesis imperfecta (AI) was present in both Fam20a and Fam20c null mice. In addition, Fam20a (-/-) mice developed disseminated calcifications of muscular arteries and intrapulmonary calcifications, similar to those of fetuin-A deficient mice, although they were normocalcemic and normophosphatemic, with normal dentin and bone. Fam20a gene expression was detected in ameloblasts, odontoblasts, and the parathyroid gland, with local and systemic effects suggesting both local and/or systemic effects for FAM20A. In contrast, Fam20c (-/-) mice lacked ectopic calcifications but were severely hypophosphatemic and developed notable lesions in both dentin and bone to accompany the AI. The bone and dentin lesions, plus the marked hypophosphatemia and elevated serum alkaline phosphatase and FGF23 levels, are indicative of autosomal recessive hypophosphatemic rickets/osteomalacia in Fam20c (-/-) mice.

Effects of alendronate on bone healing after tooth extraction in rats.

OBJECTIVES: Tooth extraction has been identified as an important risk factor for bisphosphonate-induced osteonecrosis of the jaw. Therefore, the main goal of this study was to determine the effects of alendronate on healing of the extraction socket and on interdental alveolar bone after tooth extraction in rats. MATERIALS AND METHODS: Animals were injected subcutaneously with vehicle or alendronate for 3-4 weeks before the first mandibular molar was extracted and these treatments were continued during post-extraction periods of 10, 21, 35 and 70 days. Mandibles were processed to evaluate healing of the extraction socket and adjacent alveolar bone by assessing bone formation, bone resorption and vascularity by histomorphometric techniques. RESULTS: Alendronate decreased new woven bone formation, blood vessel area, perimeter and number in the extraction socket at 10 days postextraction, but not at later time points. Furthermore, alendronate-treated rats had increased interdental alveolar bone volume and height only at 10 days postextraction. In addition, a 2.5-fold increase in the percentage of empty osteocyte lacunae was found in alveolar bone of alendronate-treated rats only at 10 days postextraction. CONCLUSIONS: Alendronate transiently decreases bone formation and vascularity in the extraction socket and delays the removal of interdental alveolar bone after tooth extraction in rats.

Skeletal effects of fibroblast growth factor mimetic (F2A) in ovariectomized rats.

The current study was designed to determine whether short-term, systemic treatment with F2A, a mimetic for FGF-2, has skeletal effects in ovariectomized (OVX) rats and adverse side effects in non-skeletal tissues. Groups of sham-operated and OVX rats were maintained untreated for 6 weeks postOVX. These groups (N=6) were then treated IP with vehicle or F2A (0.3, 1.0, or 3.0 mg/kg) daily for 14 days. Histomorphometric analyses were performed in the proximal tibial metaphyses. Hematocrit was normal in F2A-treated OVX rats. Although organ function was not evaluated, histological examination of several organs did not detect any abnormalities. F2A treatment did not increase cancellous bone mass regardless of dose, but OVX rats treated with 1 mg/kg did exhibit increased osteoclast surface. All 3 doses of F2A induced a modest increase in cancellous bone formation. Therefore, F2A appears to increase both cancellous bone resorption and formation, but these skeletal processes are in balance so that, unlike FGF-2, cancellous bone mass is not augmented. However, F2A did not induce the anemia and impaired bone mineralization associated with FGF-2. Therefore, local application of F2A for orthopedic procedures would presumably have minimal side effects, even if the peptide is released to the systemic circulation.

Osteoclast polarization and orthodontic tooth movement.

INTRODUCTION: Osteoclasts polarize when they contact activation signals that are associated with bone. Polarization is required for bone resorption and involves highly specialized mechanisms that represent attractive targets for the development of osteoclast-specific therapeutic agents. One potential use of such agents is to block tooth movement in spatially discrete locations to provide orthodontic anchorage. MATERIALS AND METHODS: Our group's research was directed toward the development of agents that inhibited the polarization of osteoclasts, and efforts were underway to develop means to experimentally modulate orthodontic tooth movement. We performed 'proof-in-principle' experiments demonstrating pharmacological blockades of orthodontic tooth movement using integrin and matrix metalloproteinase inhibitors in a rat model. RESULTS: We identified novel mechanisms underlying osteoclast bone resorption. Interactions between vacuolar H(+)-ATPase and the microfilament cytoskeleton that were unique to osteoclasts were described and characterized. Our group is now seeking to make use of this new knowledge, coupled with an emerging technique, supercomputer-based molecular modeling for the rational development of novel, osteoclast-specific therapeutic agents. CONCLUSION: Fresh insight into the molecular details of osteoclastic bone resorption provides new opportunities for identifying agents to selectively modulate osteoclast activity. Such agents may contribute to evolution of the practice of orthodontics.

Conditional expression of a Gi-coupled receptor in osteoblasts results in trabecular osteopenia.

G protein-coupled receptors (GPCRs) coupled to activation of Gs, such as the PTH1 receptor (PTH1R), have long been known to regulate skeletal function and homeostasis. However, the role of GPCRs coupled to other G proteins such as Gi is not well established. We used the tet-off system to regulate the expression of an activated Gi-coupled GPCR (Ro1) in osteoblasts in vivo. Skeletal phenotypes were assessed in mice expressing Ro1 from conception, from late stages of embryogenesis, and after weaning. Long bones were assessed histologically and by microcomputed tomography. Expression of Ro1 from conception resulted in neonatal lethality that was associated with reduced bone mineralization. Expression of Ro1 starting at late embryogenesis resulted in a severe trabecular bone deficit at 12 wk of age (>51% reduction in trabecular bone volume fraction in the proximal tibia compared with sex-matched control littermates; n = 11; P < 0.01). Ro1 expression for 8 wk beginning at 4 wk of age resulted in a more than 20% reduction in trabecular bone volume fraction compared with sex-matched control littermates (n = 16; P < 0.01). Bone histomorphometry revealed that Ro1 expression is associated with reduced rates of bone formation and mineral apposition without a significant change in osteoblast or osteoclast surface. Our results indicate that signaling by a Gi-coupled GPCR in osteoblasts leads to osteopenia resulting from a reduction in trabecular bone formation. The severity of the phenotype is related to the timing and duration of Ro1 expression during growth and development. The skeletal phenotype in Ro1 mice bears some similarity to that produced by knockout of Gs-alpha expression in osteoblasts and thus may be due at least in part to Gi-mediated inhibition of adenylyl cyclase.

Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice.

The study was designed to determine whether beta1-integrin plays a role in mediating the acute skeletal response to mechanical unloading. Transgenic (TG) mice were generated to express a dominant negative form of beta1-integrin under the control of the osteocalcin promoter, which targets expression of the transgene to mature osteoblasts. At 63 days of age, wild-type (WT) and TG mice were subjected to hindlimb unloading by tail suspension for 1 wk. Pair-fed, normally loaded WT and TG mice served as age-matched controls. Bone samples from each mouse were processed for quantitative bone histomorphometry and biomechanical testing. The skeletal phenotype of TG mice was characterized by lower cancellous bone mass in the distal femoral metaphysis (-52%) and lumbar vertebral body (-20%), reduced curvature of the proximal tibia (-20%), and decreased bone strength (-20%) and stiffness (-23%) of the femoral diaphysis with relatively normal indexes of cancellous bone turnover. Hindlimb unloading for only 1 wk induced a 10% decline in tibial curvature and a 30% loss of cancellous bone in the distal femur due to a combination of increased bone resorption and decreased bone formation in both WT and TG mice. However, the strength and stiffness of the femoral diaphysis were unaffected by short-term hindlimb unloading in both genotypes. The observed increase in osteoclast surface was greater in unloaded TG mice (92%) than in unloaded WT mice (52%). Cancellous bone formation rate was decreased in unloaded WT (-29%) and TG (-15%) mice, but, in contrast to osteoclast surface, the genotype by loading interaction was not statistically significant. The results indicate that altered integrin function in mature osteoblasts may enhance the osteoclastic response to mechanical unloading but that it does not have a major effect on the development of cancellous osteopenia in mice during the early stages of hindlimb unloading.

A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats.

Previous studies have shown that parathyroid hormone (PTH) stimulates bone formation and completely restores lost cancellous bone at skeletal sites with moderate osteopenia in relatively young ovariectomized (OVX) rats. The current study was designed to determine whether PTH has similar bone anabolic effects in aged OVX rats and to compare the bone restorative response to PTH at skeletal sites with moderate and severe osteopenia. Female Sprague-Dawley rats were subjected to sham surgery or bilateral ovariectomy at 3 months of age and maintained untreated for the first year after surgery to allow for the development of moderate vertebral osteopenia and severe tibial osteopenia in OVX rats. Groups of baseline control and OVX rats were sacrificed at the end of this pretreatment period. The remaining OVX rats were then treated for 10 weeks with vehicle, antiresorptive agents alone (estrogen, the bisphosphonate risedronate, or calcitonin) or PTH alone. Other groups of OVX rats were treated concurrently with PTH and each of the antiresorptive agents. As expected, the proximal tibia of baseline OVX rats exhibited severe cancellous osteopenia, whereas the first lumbar vertebral body was moderately osteopenic. Treatment of OVX rats with antiresorptive agents alone failed to restore cancellous bone at both skeletal sites, whereas treatment with PTH alone markedly stimulated bone formation and completely restored lost cancellous bone in the lumbar vertebra. PTH also stimulated bone formation and in the severely osteopenic proximal tibia of OVX rats but only marginally restored lost cancellous bone, possibly due to an inadequate number of bone spicules to serve as a foundation for new bone formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats.

The goal of this study is to determine whether the previously observed, short-term protective effect of estrogen and diphosphonate compounds against osteopenia in ovariectomized (OVX) rats can be maintained for an entire year. Sham-operated control and OVX rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and risedronate (NE-58095) for 360 days after surgery. Their proximal tibiae and first lumbar vertebrae were processed undecalcified for quantitative bone histomorphometry. Both skeletal sites in vehicle-treated OVX rats were characterized by decreased cancellous bone volume and increases in most cellular and fluorochrome-based indices of bone formation and resorption. Treatment of OVX rats with estrogen or diphosphonate compounds depressed bone turnover and provided nearly complete protection against cancellous bone loss. Long-term EHDP treatment induced a moderate mineralization defect, as indicated by increased absolute osteoid volume and a high proportion of osteoid surfaces devoid of adjacent osteoblasts. In contrast, NE-58095 had minimal effects on bone mineralization. These findings indicate that diphosphonate compounds and estrogen provide long-term protection against tibial and vertebral osteopenia in OVX rats. They further indicate that diphosphonate compounds merit consideration as an alternative to estrogen for the prevention of postmenopausal bone loss.

Sequential treatment with basic fibroblast growth factor and parathyroid hormone restores lost cancellous bone mass and strength in the proximal tibia of aged ovariectomized rats.

This study was designed to determine whether sequential treatment with basic fibroblast growth factor (bFGF) and parathyroid hormone (PTH) can restore lost cancellous bone mass and strength at a severely osteopenic skeletal site in aged ovariectomized (OVX) rats. Female Sprague-Dawley rats were subjected to sham surgery or ovariectomy at 3 months of age and maintained untreated for the first year after surgery. At 15 months of age, groups of baseline control and OVX rats were killed and catheters were inserted in the jugular veins of all remaining rats. Two groups of OVX rats were injected intravenously (iv) daily with bFGF for 14 days at a dose of 200 microg/kg body weight. At the end of bFGF treatment, one group was killed whereas the other group was subjected to 8 weeks of treatment with synthetic human PTH 1-34 [hPTH(1-34)] consisting of subcutaneous (sc) injections 5 days/week at a dose of 80 microg/kg. Another group of OVX rats was treated iv with vehicle for 2 weeks followed by treatment with PTH alone for 8 weeks. Other groups of sham-operated control rats and OVX rats were treated iv and sc with vehicle alone. The right proximal tibia from each rat was processed undecalcified for quantitative bone histomorphometry and the left proximal tibia was subjected to biomechanical testing. Baseline and vehicle-treated OVX rats were severely osteopenic because their tibial cancellous bone volumes were less than 5% compared with mean values of 20.3% and 15.0% in baseline and vehicle-treated control rats, respectively. Treatment of OVX rats for 2 weeks with bFGF alone did not significantly increase tibial cancellous bone volume but induced marked increases in osteoid volume, osteoblast surface, and osteoid surface. Sequential treatment of aged OVX rats with bFGF and PTH increased tibial cancellous bone volume (15.1%) and load to failure to at least the level of vehicle-treated control rats. Tibial cancellous bone volume (10.8%) and load to failure also were significantly increased by treatment with PTH alone, and these variables were not significantly different from those of OVX rats treated with bFGF + PTH. However, tibial ash density was significantly greater in OVX rats treated sequentially with bFGF and PTH compared with OVX rats treated with PTH alone. Our findings suggest that sequential treatment with bFGF and PTH may be useful for restoration of lost cancellous bone in the severely osteopenic, estrogen-deplete skeleton, but it cannot be concluded with certainty that this sequential treatment has a greater bone restorative effect than treatment with PTH alone.

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.

Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats.

Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham surgery (control). Groups of ovariectomized (OVX) and control rats were injected daily with low, medium, or high doses of 17 beta-estradiol (10, 25, or 50 micrograms/kg BW, respectively). An additional group of OVX and control rats was injected daily with vehicle alone. All rats were killed 35 days after OVX, and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Trabecular bone volume was markedly reduced in vehicle-treated OVX rats relative to that in control rats (12.1% vs. 26.7%). This bone loss was associated with a 2-fold increase in osteoclast surface and a 4-fold increase in osteoblast surface. The bone formation rate, studied with fluorochrome labeling, was also significantly elevated in vehicle-treated OVX rats (0.111 vs. 0.026 micron3/micron2.day). In contrast, treatment of OVX rats with the three doses of estradiol resulted in normalization of tibial trabecular bone volume and a decline in histomorphometric indices of bone resorption and formation. Our results indicate that estrogen treatment provides complete protection against osteopenia in OVX rats. The protective mechanism involves estrogenic suppression of bone turnover. These findings are consistent with the skeletal effects of estrogen therapy in postmenopausal women.

The positive effect of parathyroid hormone on femoral neck bone strength in ovariectomized rats is more pronounced than that of estrogen or bisphosphonates.

This study elucidates the effect of PTH, estrogen, and the bisphosphonate Risedronate (NE-58095) on femoral neck bone strength in ovariectomized (OVX) rats aged 90 days at the beginning of the investigation. Furthermore, the effects of these monotherapies were compared with those of concurrent treatment with PTH plus estrogen or PTH plus bisphosphonate. Four weeks after surgery the rats were randomized into a sham-operated vehicle-treated group, an OVX vehicle-treated group, and the various treatment groups and followed for 5 and 15 weeks. The proximal one-third of the left femur was then subjected to geometrical measurements and biomechanical testing. Neither ovariectomy nor the different treatment regimens influenced femoral neck geometry. OVX rats exhibited a decrease in femoral neck bone strength compared with control rats. This was most evident after 5 weeks. Treatment of OVX rats with Risedronate or estrogen alone tended to increase bone strength to control level, though these findings were nonsignificant. In contrast, treatment with PTH showed a highly significant increase in femoral neck biomechanical competence. Concurrent treatment with PTH plus estrogen or PTH plus Risedronate also significantly increased the femoral neck bone strength, but neither showed any advantage over treatment with PTH alone. It is concluded that treatment with PTH increases the strength of the femoral neck in estrogen-depleted rats in a highly significant manner, and that this effect is much more pronounced than the effect of the two antiresorptive agents estrogen or Risedronate. Thus, these findings provide further support for the anabolic effect of PTH and add weight to the argument for the promising potential of PTH in the treatment of postmenopausal osteoporosis.

Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats.

The study was designed to compare the therapeutic efficacy of estrogen, the bisphosphonate risedronate (NE-58095), and PTH for restoration of lost bone mass in osteopenic, ovariectomized (OVX) rats. In addition, the skeletal effects of these single treatments were compared to those of concurrent treatments with PTH + estrogen or PTH + NE-58095. OVX rats were untreated for the first 4 weeks postovariectomy to allow for the development of moderate tibial osteopenia. These animals were then subjected to the various treatments for periods of 5, 10, and 15 weeks. Their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Treatment of osteopenic OVX rats with estrogen or NE-58095 alone depressed bone turnover and prevented additional cancellous bone loss from occurring during the treatment period. However, these therapeutic agents failed to restore lost bone in OVX rats to control levels. In contrast, OVX rats treated with PTH alone exhibited a marked stimulation of bone formation which resulted in augmentation of cancellous bone mass to a level 2-fold greater than that of vehicle-treated control rats. Concurrent treatment of OVX rats with PTH + estrogen as well as PTH + NE-58095 also effectively reversed cancellous osteopenia in OVX rats, but did not appear to be more beneficial to the estrogen-deplete skeleton than treatment with PTH alone. The results indicate that PTH is a powerful stimulator of bone formation and completely restores lost cancellous bone in osteopenic OVX rats. Furthermore, the bone anabolic effects of PTH are much more pronounced than those of estrogen or bisphosphonates. These findings in an animal model of estrogen depletion provide support for PTH as a potentially effective treatment for oophorectomized and postmenopausal women with established osteoporosis.

Chronic administration of 1,25-dihydroxyvitamin D3: increased bone but impaired mineralization.

Male Sprague-Dawley rats were infused continuously for 13 days with vehicle or 75 pmol (31.2 ng)/day 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by means of Alzet osmotic minipumps implanted s.c. Animals infused with 1,25-(OH)2D3 exhibited mild hypercalcemia (11.2 vs. 10.2 mg/dl in controls), a 136% increase in the serum concentration of 1,25-(OH)2D3 (187 vs. 79 pg/ml), and a 59% decrease in serum 25-hydroxyvitamin D (12 vs. 29 ng/ml). The proximal tibial metaphysis of these animals was characterized by increased trabecular bone volume (15% vs. 6.5%), osteoid accumulation (4.2% vs. 0.1%), increased osteoblast surface and number (31% and 19/mm vs. 21% and 14/mm, respectively), and decreased osteoclast surface and number (11% and 2/mm vs. 36% and 6/mm, respectively). Similar but less striking changes were seen in the lumbar vertebra. Increases in the fat-free weight and calcium content of the tibia and lumbar vertebra were consistent with the increase in trabecular bone volume after 1,25-(OH)2D3 infusion. However, tetracycline labeling and the incorporation of 45Ca in these bones were reduced by 1,25-(OH)2D3 infusion. In addition, mineralization lag time was prolonged in the lumbar vertebrae of 1,25-(OH)2D3-infused rats. Our findings indicate that chronic 1,25-(OH)2D3 administration increases bone mass, but at the cost of impaired bone mineralization.

Endocrine and pharmacological suppressors of bone turnover protect against osteopenia in ovariectomized rats.

This study was designed to test the hypothesis that endocrine and pharmacological suppressors of bone turnover prevent the development of osteopenia during estrogen deficiency. Sham-operated control and ovariectomized (OVX) rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and NE-58095 [2-(3-pyridinyl)2-hydroxyethylidene-1,1-bisphosphonate disodium] for 35 or 70 days after surgery. Their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Vehicle-treated OVX rats were characterized by decreased cancellous bone volume and 3- to 4-fold increases in osteoblast surface, osteoclast surface, bone formation rate, and bone resorption rate. Treatment of OVX rats with estrogen and NE-58095 provided complete protection against bone loss and significantly depressed all of the above indices of bone turnover. OVX rats treated with EHDP exhibited at least partial protection against bone loss and decreased bone turnover. EHDP induced a mild mineralization defect, as indicated by a prolonged mineralization lag time at the tibial endocortical surface. The new diphosphonate compound NE-58095 did not impair bone mineralization. Our results indicate that endocrine and pharmacological suppressors of bone turnover prevent the development of osteopenia during the early stages of estrogen deficiency. If confirmed by clinical trials in humans, diphosphonate compounds may prove to be an alternative to estrogen for the prevention of postmenopausal bone loss.

Sequential treatment with basic fibroblast growth factor and PTH is more efficacious than treatment with PTH alone for increasing vertebral bone mass and strength in osteopenic ovariectomized rats.

The study was designed 1) to determine whether treatment with basic fibroblast growth factor (bFGF) and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic ovariectomized rats, and 2) to assess whether prior and concurrent administration of the antiresorptive agents estrogen and risedronate suppresses the bone anabolic response to treatment with bFGF alone and sequential treatment with bFGF and PTH. Three-month-old female Sprague Dawley rats were ovariectomized (OVX) or sham-operated (sham) and maintained untreated for 1 yr. Baseline sham and OVX rats were killed at this time (15 months of age). Groups of rats were injected sc with estrogen (10 microg/kg, 4 d/wk), risedronate (5 microg/kg, 2 d/wk), or vehicle. At the end of the second week of antiresorptive treatment, catheters were inserted into the jugular veins of all rats, and vehicle or bFGF at a dose of 250 microg/kg was injected daily for 14 d. Three groups of rats were killed at the end of bFGF treatment. The remaining rats were continued on their respective antiresorptive therapy and injected sc with vehicle or synthetic human PTH-(1-34) at a dose of 80 microg/kg, 5 d/wk, for 8 wk. Lumbar vertebrae were processed for cancellous bone histomorphometry and biomechanical testing. Ovariectomy resulted in a decrease in vertebral bone mass and strength. Treatment of OVX rats for 14 d with bFGF markedly increased osteoblast surface, osteoid surface, and osteoid volume compared with vehicle treatment of sham and OVX rats. Furthermore, osteoid bridges were observed extending between preexisting trabeculae in bFGF-treated OVX rats. Prior and concurrent administration of estrogen and risedronate did not suppress these bone anabolic effects of bFGF. Treatment of OVX rats with PTH alone increased vertebral cancellous bone mass and strength to the level of vehicle-treated sham rats. Sequential treatment of OVX rats with bFGF and PTH further augmented vertebral bone mass and strength to a level above that observed in OVX rats treated with PTH alone. The improvements in bone mass and strength were associated with an increase in trabecular thickness in OVX rats treated with PTH alone and with an increase in trabecular thickness and node to terminus ratio, an index of trabecular connectivity, in OVX rats treated sequentially with bFGF and PTH. Cotreatment with estrogen and risedronate did not suppress the anabolic response of bone to bFGF and PTH. In fact, a trend for an even greater increase in cancellous bone mass and node to terminus ratio was observed in OVX rats treated with risedronate, bFGF, and PTH. These findings indicate that sequential treatment with bFGF and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic OVX rats.

Orchiectomy markedly reduces the concentration of the three isoforms of transforming growth factor beta in rat bone, and reduction is prevented by testosterone.

Available evidence indicates that transforming growth factor beta (TGFbeta) is produced by bone cells, that production is enhanced by testosterone and dihydrotestosterone, and that TGFbeta is an important modulator of bone formation, induction, and repair. To determine the relative concentrations of isoforms of skeletal TGFbeta, whether orchiectomy alters the concentration of TGFbeta in long bones, and whether alteration is prevented by testosterone replacement, male Sprague-Dawley rats were either sham-operated and given placebo (n = 20) or orchiectomized and given either placebo (n = 20) or 100 mg testosterone (n = 20) by slow-release pellets implanted sc at the back of the neck and killed at 6 weeks. Orchiectomy did not change serum calcium and lowered serum testosterone and serum phosphorus; these reductions were prevented by testosterone replacement. TGFbeta1 in skeletal extracts was much more abundant than TGFbeta2 or TGFbeta3. Orchiectomy reduced skeletal TGFbeta by over 80 percent, and reduction was prevented by testosterone replacement. The relative abundance of the three isoforms of TGFbeta in bone was not influenced by orchiectomy or testosterone replacement, and skeletal messenger RNA of TGFbeta1 and TGFbeta2 was not altered 4 weeks after orchiectomy. Messenger RNA for TGFbeta3 was below the limits of detection. Thus, testosterone deficiency markedly diminishes skeletal TGFbeta, and reduction is prevented by testosterone replacement. The findings support the hypothesis that testosterone and TGFbeta are required for maintenance of the skeleton in male rats.

The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading.

Skeletal unloading results in osteopenia. To examine the involvement of vitamin D in this process, the rear limbs of growing rats were unloaded, and alterations in bone calcium and bone histology were related to changes in serum calcium (Ca), inorganic phosphorus, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D [24,25-(OH)2D], and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Acute skeletal unloading induced a transitory inhibition of Ca accumulation in unloaded bones. This was accompanied by a transitory rise in serum Ca, a 21% decrease in longitudinal bone growth (P less than 0.01), a 32% decrease in bone surface lined with osteoblasts (P less than 0.05), no change in bone surface lined with osteoclasts, and a decrease in circulating 1,25-(OH)2D from 130 +/- 10 to 53 +/- 11 pg/ml. No significant changes in the serum concentrations of inorganic phosphorus, 25-hydroxyvitamin D, or 24,25-(OH)2D were observed. After 2 weeks of unloading, bone Ca stabilized at approximately 70% of control values, and serum Ca and 1,25-(OH)2D returned to control values. Maintenance of a constant serum 1,25-(OH)2D concentration by chronic infusion of 1,25-(OH)2D (Alza osmotic minipump) throughout the study period did not prevent the bone changes induced by acute unloading. These results suggest that acute skeletal unloading in the growing rat produces a transitory inhibition of bone formation, which, in turn, produces a transitory hypercalcemia, leading to a temporary decrease in serum 1,25-(OH)2D. No evidence could be found for a direct involvement of 1,25-(OH)2D in the bone changes induced by skeletal unloading.