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.     

Teriparatide [rhPTH (1-34)], but not strontium ranelate, demonstrated bone anabolic efficacy in mature, osteopenic, ovariectomized rats

We compared teriparatide (TPTD) and strontium ranelate (SR) efficacy on bone formation activity in a mature rat model of estrogen-deficiency bone loss. Rats were ovariectomized (OVX) at age 6 months and permitted to lose bone for 2 months to establish osteopenia before initiation of treatment with TPTD (5 or 15 μg/kg · d sc) or SR (150 or 450 mg/kg · d oral gavage). After 3 wk, RT-PCR analyses of bone formation genes in the distal femur metaphysis showed significant elevation of collagen 1α2, osteocalcin, bone sialoprotein, alkaline phosphatase, and Runx2 gene expression at both TPTD doses, relative to OVX controls. SR had no significant effect on expression of these genes. TPTD treatment for 12 wk dose dependently increased lumbar vertebral (LV) and femoral midshaft bone mineral content (BMC) and bone mineral density over pretreatment and age-matched OVX controls. SR 150 increased BMC, and SR 450 increased BMC and bone mineral density of femoral midshaft and LV over OVX controls. There were significant dose-dependent TPTD increases of LV and femoral neck strength, and TPTD 15 also increased midshaft strength compared with pretreatment and age-matched OVX controls. SR did not enhance bone strength relative to pretreatment or age-matched OVX controls. Histomorphometry of the proximal tibial metaphysis showed dose-dependent effects of TPTD on trabecular area, number, width, and osteoblast surface, bone mineralizing surface, and bone formation rate relative to pretreatment and age-matched OVX controls, whereas SR had no effect on these parameters. These findings confirmed the bone anabolic efficacy of teriparatide, but not SR in mature, osteopenic, OVX rats.     

Long-term treatment of lasofoxifene preserves bone mass and bone strength and does not adversely affect the uterus in ovariectomized rats

The purpose of this study was to determine the long-term effects of lasofoxifene, a new selective estrogen receptor modulator, on bone mass, bone strength, and reproductive tissues in ovariectomized (OVX) rats. Sprague Dawley female rats at 3.5 months of age were OVX and treated orally with lasofoxifene (60, 150, or 300 microg/kg x d) for 52 wk. The urinary deoxypyridinoline/creatinine ratio was significantly lower in all lasofoxifene-treated OVX rats compared with OVX controls at wk 26. Peripheral quantitative computerized tomography analysis of proximal tibial metaphysis showed that the significant loss in trabecular content and density induced by OVX was significantly prevented by lasofoxifene treatment. Proximal tibial and lumber vertebral trabecular bone histomorphometric analysis showed that all doses of lasofoxifene significantly reduced OVX-induced bone loss by decreasing bone resorption and bone turnover. The ultimate strength, energy, and toughness of the fourth lumbar vertebral body in OVX rats treated with all doses of lasofoxifene were significantly higher compared with those in OVX controls, and did not differ significantly from those in sham controls. Uterine weight in OVX rats treated with lasofoxifene was slightly, but significantly, higher when compared with that in OVX controls, but was still much less than that in sham controls. No abnormal finding associated with lasofoxifene was observed with uterine histology examination. In summary, long-term treatment with lasofoxifene preserves bone mass and bone strength and does not adversely affect the uterus in OVX rats. These data suggest that lasofoxifene is an effective antiosteoporosis agent, and its efficacy and safety can be maintained over an extended period of time.     

Sevelamer restores bone volume and improves bone microarchitecture and strength in aged ovariectomized rats

Sevelamer hydrochloride, a noncalcium phosphate binder, has been shown to reduce coronary artery and aortic calcification, and to improve trabecular bone mineral density in hemodialysis patients with chronic kidney disease. Here, we examined whether sevelamer given orally for 12 wk with normal food could restore bone volume (BV) and strength in aged ovariectomized (OVX) rats starting at 4 wk after OVX. Dual-energy x-ray absorptiometry, microcomputerized tomography, and bone histomorphometry analyses showed that OVX animals receiving sevelamer had increased trabecular BV (51%), trabecular number (43%), trabecular thickness (9%), cortical thickness (16%), mineral apposition rate (103%), bone formation rate (25%), and enhanced cortical and trabecular bone mechanical strength as compared with OVX rats. Sevelamer decreased collagen C telopeptide, increased osteocalcin levels, and decreased phosphate and magnesium levels without affecting calcium levels in the blood. Although sevelamer was not absorbed systemically, it stimulated osteoblast differentiation in BM-derived mesenchymal stem cell cultures, as evaluated by alkaline phosphatase positive colony-forming units, and inhibited recombinant human soluble receptor activator of nuclear factor-kappaB ligand-induced osteoclast differentiation, as evaluated by tartrate-resistant acid phosphatase positive cells in bone mineral-hematopoietic stem cell cultures. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis revealed that 69 proteins were differently expressed after OVX, of which 30% (20 of 69) were reversed to sham activity after sevelamer intake. PTH, fibroblast growth factor-23, and cytokine profile in serum were not significantly changed. Together, these results suggest that sevelamer in food increases the BV and improves biomechanical properties of bone in OVX rats.     

Human parathyroid hormone-(1-34) increases bone mass in ovariectomized and orchidectomized rats

In intact growing rats, intermittent administration of low doses of PTH increases bone mass. As gonadal hormones are considered to be essential for normal bone growth, the anabolic effect of PTH may be mediated or modified by these hormones. The objective of this research was to determine if the anabolic effect of PTH would be altered in female ovariectomized (OVX) and male orchidectomized (ORCHX) rats. Two weeks after ovariectomy, orchidectomy, or sham operations, 5-week-old rats (eight per group) were given daily sc injections of human PTH (1-34) (8 micrograms/100 g) or vehicle. After 12 days of treatment, all rats were killed; castration was confirmed, and sera, femurs, tibias, and kidneys were collected. Calcium (Ca) and dry weight (DW) of trabecular and cortical bone of distal half-femurs were measured. Female OVX rats were osteopenic compared to their sham-operated controls, as the bone mass of distal femurs decreased while body weight increased. In PTH-treated females, total bone Ca and DW per 100 g BW increased significantly by 16% and 21%, respectively, in sham-operated rats and by 21% and 25%, respectively, in OVX rats compared to the appropriate control values. ORCHX rats were also osteopenic, as the bone mass of distal femurs was significantly decreased compared to that in sham-operated males. However, as body weight also decreased, the bone mass per unit BW was not altered. In PTH-treated males, total bone Ca and DW per 100 g BW increased significantly by 34% and 25%, respectively, in sham-operated rats by 32% and 29%, respectively, in ORCHX rats compared to their appropriate control values. Serum Ca, creatinine, and alkaline phosphatase levels were normal and comparable in all rats. We conclude that PTH increased bone mass in control, OVX, and ORCHX rats, and the anabolic response to PTH is not dependent on gonadal hormones.     

Binge alcohol treatment increases vertebral bone loss following ovariectomy: compensation by intermittent parathyroid hormone

BACKGROUND: Postmenopausal estrogen deficiency and alcohol abuse are known risk factors for osteoporosis. Previous studies of the combined effect of alcohol and ovariectomy on bone loss using chronic alcohol-feeding models have not demonstrated additional alcohol-induced bone loss in ovariectomized (OVX) animals. Binge alcohol treatment causes rapid bone loss in male rats. We hypothesized that binge alcohol would cause additional bone loss in OVX rats. METHODS: Ninety-six adult (400 g) female Sprague-Dawley rats (48 sham-operated and 48 OVX, pair fed) were randomly divided into 4 treatment groups: (a) saline-treated, (b) binge alcohol-treated (3 g/kg alcohol as a 20% weight to volume alcohol/saline solution, intraperitoneal (IP), 3 times per week), (c) parathyroid hormone (PTH)-treated (80 microg/kg, SC, 5 d/wk), and (d) binge alcohol plus PTH. Rats were treated for either 2 or 4 weeks. Following treatment periods, blood was collected for alcohol concentration (BAC) measurements; lumbar vertebrae were removed for bone mineral density (BMD) levels, trabecular microarchitecture assessment, and vertebral compressive strength analysis. RESULTS: Peak binge BACs averaged 300 mg/dL. Alcohol and OVX decreased cancellous BMD: alcohol and OVX treatment in combination caused additional cancellous BMD loss and significant cortical BMD reductions. Compressive strength was also decreased by OVX and alcohol. Combination treatment resulted in further declines in bone strength. Micro-CT analysis revealed a significant effect of combined OVX and alcohol treatment resulting in decreased trabecular bone volume/total volume (BV/TV). Intermittent PTH administration compensated for losses of BMD, compressive strength, and restored BV/TV deficits caused by OVX, alcohol, or their combination. CONCLUSIONS: Bone loss following OVX can be significantly increased by concurrent binge alcohol treatment. The effects of alcohol and OVX are compensated by concurrent intermittent treatment with PTH. These results suggest that postmenopausal women who abuse alcohol may place their skeleton at additional risk for osteoporotic fracture.     

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.     

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.     

The effects of combination of alendronate and human parathyroid hormone(1-34) on bone strength are synergistic in the lumbar vertebra and additive in the femur of C57BL/6J mice

A cyclic PTH regimen is as effective as a daily regimen on bone density gain in humans and in improving bone quality in mice. Our previous murine study evaluated the effects of a cyclic PTH regimen in the absence of a bisphosphonate, whereas our human study addressed the effects of a cyclic PTH regimen in the presence of ongoing alendronate (ALN) treatment. Accordingly, the current study examined the effects of cyclic or daily PTH regimens in combination with ALN on bone quality and bone density in mice. Twenty-week-old, female C57BL/6J mice were treated with the following sc injections (n = 10): 1) vehicle for 5 d/wk (control); 2) ALN (20 microg/kg x d) 3 d/wk (ALN); 3) human PTH(1-34) (40 microg/kg x d) 5 d/wk (daily PTH); 4) daily PTH in addition to ALN (daily PTH plus ALN); 5) PTH 5 d/wk and vehicle 5 d/wk alternating weekly (cyclic PTH); 6) cyclic PTH in addition to ALN (cyclic PTH plus ALN); and 7) PTH and ALN alternating weekly (alt PTH and ALN). Bone mineral density was measured weekly by dual-energy x-ray absorptiometry, and at 7 wk, bone markers, bone structure, and bone strength were evaluated by biochemical assays, peripheral quantitative computed tomography and mechanical testing, respectively. At 7 wk, all treatments significantly increased femoral and vertebral bone mineral density. ALN slightly decreased endosteal circumference, whereas PTH increased periosteal circumference, resulting in significant increases in femoral cortical thickness in all groups. PTH and ALN enhanced bone strength synergistically in the lumbar vertebrae and additively in the femur. Combined therapy, however, had no effects on bone markers. The results show that combinations of ALN and PTH, in both daily and cyclic regimens, produce more beneficial effects than treatment with either agent alone, suggesting that the mechanisms of actions of ALN and PTH on bone quality may be complementary.     

Combination treatment with pioglitazone and fenofibrate attenuates pioglitazone-mediated acceleration of bone loss in ovariectomized rats

Peroxisome proliferator-activated receptor (PPAR) γ agonists, such as pioglitazone (Pio), improve glycemia and lipid profile but are associated with bone loss and fracture risk. Data regarding bone effects of PPARα agonists (including fenofibrate (Feno)) are limited, although animal studies suggest that Feno may increase bone mass. This study investigated the effects of a 13-week oral combination treatment with Pio (10?mg/kg per day)+Feno (25?mg/kg per day) on body composition and bone mass parameters compared with Pio or Feno alone in adult ovariectomized (OVX) rats, with a 4-week bone depletion period, followed by a 6-week treatment-free period. Treatment of OVX rats with Pio+Feno resulted in ～50% lower fat mass gain compared with Pio treatment alone. Combination treatment with Pio+Feno partially prevented Pio-induced loss of bone mineral content (～45%) and bone mineral density (BMD; ～60%) at the lumbar spine. Similar effects of treatments were observed at the femur, most notably at sites rich in trabecular bone. At the proximal tibial metaphysis, concomitant treatment with Pio+Feno prevented Pio exacerbation of ovariectomy-induced loss of trabecular bone, resulting in BMD values in the Pio+Feno group comparable to OVX controls. Discontinuation of Pio or Feno treatment of OVX rats was associated with partial reversal of effects on bone loss or bone mass gain, respectively, while values in the Pio+Feno group remained comparable to OVX controls. These data suggest that concurrent/dual agonism of PPARγ and PPARα may reduce the negative effects of PPARγ agonism on bone mass.     

去卵巢大鼠椎体松质骨骨小梁微结构改变及其对生物力学的影响

目的观察骨质疏松和正常状态下椎体松质骨的微观结构改变,分析其对骨生物力学的影响。方法将12只4月龄雌性Lewis大鼠随机分为去卵巢组(OVX)组和假手术组(Sham),每组各6只。OVX组行双侧卵巢切除术,假手术组仅显露双侧卵巢。术后6个月处死动物,取尾椎(L_(4-7))行Micro-CT分析及生物力学测试。结果去卵巢6个月后,OVX组大鼠体积骨密度(vBMD)和组织骨密度(tBMD)较Sham组显著降低,松质骨骨小梁的骨体积分数(BV/TV)和数目(TB.N)都明显低于Sham组,骨小粱表面积密度(BS/BV)、结构模拟指数(SMI)和间距(Tb.Sp)显著高于Sham组,差异有统计学意义。但2组间骨小梁厚度(Tb.Th)差异无统计学意义。生物力学测试结果表明,去势6个月后,OVX组大鼠骨质生物力学性能显著下降。骨小梁力学性能与骨小梁体积分数(Adjusted R~2=0.750和数目(Adjusted R~2=0.861)呈正线性相关,而与结构模拟指数(Adjusted R~2=0.716)和骨小梁间距(Adjusted R~2=0.830)呈负线性相关。结论松质骨骨小梁微观结构的改变可影响骨质的生物力学性能,二者之间具有一定的线性关系。     

去卵巢大鼠骨形成参数和血清碱性磷酸酶的相关性研究

目的　观察去卵巢大鼠骨组织形态计量学参数和血清碱性磷酸酶 (ALP)之间的相关性。方法　 4个半月龄 SD大鼠双侧卵巢去除术后预防用药 90 d。用骨组织形态计量学方法测定大鼠胫骨组织近端松质骨形态计量学参数 ,并测定血清 ALP含量。结果　去卵巢组大鼠血清 ALP含量增加 ,骨形成参数增加 (P<0 .0 5)。去卵巢组和预防用药组骨形成参数与 ALP测量值之间有相关性 (P<0 .0 5)。去卵巢组的骨组织形态计量学参数与 ALP测量值之间的相关系数大于预防用药组。结论　去卵巢大鼠血清 ALP与骨形成参数之间存在相关性 ,这种相关性在给药后下降。     

Insulin receptor substrate-1 is required for bone anabolic function of parathyroid hormone in mice

Bone anabolic action of PTH has been suggested to be mediated by induction of IGF-I in osteoblasts; however, little is known about the molecular mechanism by which IGF-I leads to bone formation under the PTH stimulation. This study initially confirmed in mouse osteoblast cultures that PTH treatment increased IGF-I mRNA and protein levels and alkaline phosphatase activity, which were accompanied by phosphorylations of IGF-I receptor, insulin receptor substrate (IRS)-1 and IRS-2, essential adaptor molecules for the IGF-I signaling. To learn the involvement of IRS-1 and IRS-2 in the bone anabolic action of PTH in vivo, IRS-1-/- and IRS-2-/- mice and their respective wild-type littermates were given daily injections of PTH (80 mug/kg) or vehicle for 4 wk. In the wild-type mice, the PTH injection increased bone mineral densities of the femur, tibia, and vertebrae by 10-20% without altering the serum IGF-I level. These stimulations were similarly seen in IRS-2-/- mice; however, they were markedly suppressed in IRS-1-/- mice. Although the PTH anabolic effects were stronger on trabecular bones than on cortical bones, the stimulations on both bones were blocked in IRS-1-/- mice but not in IRS-2-/- mice. Histomorphometric and biochemical analyses showed an increased bone turnover by PTH, which was also blunted by the IRS-1 deficiency, though not by the IRS-2 deficiency. These results indicate that the PTH bone anabolic action is mediated by the activation of IRS-1, but not IRS-2, as a downstream signaling of IGF-I that acts locally as an autocrine/paracrine factor.     

Subcutaneous administration of insulin-like growth factor (IGF)-II/IGF binding protein-2 complex stimulates bone formation and prevents loss of bone mineral density in a rat model of disuse osteoporosis.

Elevated serum levels of insulin-like growth factor binding protein-2 (IGFBP-2) and a precursor form of IGF-II are associated with marked increases in bone formation and skeletal mass in patients with hepatitis C-associated osteosclerosis. In vitro studies indicate that IGF-II in complex with IGFBP-2 has high affinity for bone matrix and is able to stimulate osteoblast proliferation. The purpose of this study was to determine the ability of the IGF-II/IGFBP-2 complex to increase bone mass in vivo. Osteopenia of the femur was induced by unilateral sciatic neurectomy in rats. At the time of surgery, 14-day osmotic minipumps containing vehicle or 2 microg IGF-II+9 microg IGFBP-2/100g body weight/day were implanted subcutaneously in the neck. Bone mineral density (BMD) measurements were taken the day of surgery and 14 days later using a PIXImus small animal densitometer. Neurectomy of the right hindlimb resulted in a 9% decrease in right femur BMD (P<0.05 vs. baseline). This loss in BMD was completely prevented by treatment with IGF-II/IGFBP-2. On the control limb, there was no loss of BMD over the 14 days and IGF-II/IGFBP-2 treatment resulted in a 9% increase in left femur BMD (P<0.05). Bone histomorphometry indicated increases in endocortical and cancellous bone formation rates and in trabecular thickness. These results demonstrate that short-term administration of the IGF-II/IGFBP-2 complex can prevent loss of BMD associated with disuse osteoporosis and stimulate bone formation in adult rats. Furthermore, they provide proof of concept for a novel anabolic approach to increasing bone mass in humans with osteoporosis.     

柚皮苷联合运动对去势大鼠骨量和骨强度的影响

目的观察柚皮苷（NG）联合中强度跑台运动对去势大鼠骨质疏松模型的治疗效果。方法2月龄雌性SD大鼠80只,用随机数字法分为假手术组（SHAM）、去势组（OVX）、去势＋不同浓度（40、100、200 mg/kg）柚皮苷组（OVX＋NG）、去势＋跑台＋柚皮苷组（OVX＋EX＋NG）、去势＋跑台组（OVX＋EX）、雌激素组（OVX＋E2）,每组10只。大鼠切除卵巢2个月后开始给药,给药60 d后观察各组大鼠股骨骨密度（BMD）,Micro-CT参数,血清生化指标及及股骨力学性能与组织学改变。结果柚皮苷与跑台运动干预60 d后,OVX＋EX＋NG组大鼠股骨颈力学强度、股骨BMD、BV/TV、Tb.N、Tb.Th等均高于单纯去势组（P〈0.05）,OVX＋EX＋NG组治疗效果与柚皮苷浓度呈正相关,以200 mg/kg柚皮苷效果最佳;200 mg/kg柚皮苷联合中强度跑台运动可进一步提高治疗效果。OVX＋NG组大鼠血清骨钙素升高,Ⅰ型胶原C端肽（CTX-1）降低;OVX＋EX组大鼠骨钙素与CTX-1均降低;OVX＋EX＋NG组大鼠骨钙素水平与柚皮苷组（200 mg/kg）无差异,但高于OVX＋EX。OVX＋EX＋NG组CTX-1水平低于单纯柚皮苷组和单纯跑台组（P〈0.05）。结论柚皮苷联合中强度跑台运动可提高去势大鼠股骨BMD,增加骨小梁数目,改善骨代谢指标,提高股骨力学性能。     

Comparison of the anabolic effects of synthetic parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34 on bone in rats

The objective of this study was to determine whether intermittent synthetic human PTH-related protein (PTHrP 1-34) will mimic the anabolic effect of PTH and increase bone mass in rats. Dose response experiments were done on young, male Sprague-Dawley rats given sc vehicle, human (h) PTH (1-34) at 8 micrograms/100 g or PTHrP (1-34) at 1-32 micrograms/100 g daily for 12 days or 26 days. On the last day, 3 h after injections, rats were killed and serum, femurs, and tibias harvested. Trabecular and cortical bone of distal half femurs were analyzed for calcium (Ca) and hydroxyproline content and dry weight. Tibia metaphyseal bone was analyzed using conventional histomorphometry techniques. Our results showed that low doses of PTHrP (1-34) did not increase bone mass or bone forming surfaces. After 12 days, PTH, at 8 micrograms/100 g, increased trabecular Ca, dry weight, and hydroxyproline by approximately 19%, 36%, and 53%, respectively, while the bone mass of PTHrP-treated rats was comparable to vehicle-treated rats. PTHrP at a higher dose of 32 micrograms/100 g, increased trabecular bone mass by 30-37%, compared to the 43-48% increase induced by PTH at 8 micrograms/100 g after 12 days. When treatment was extended to 26 days, PTHrP, at 16 micrograms/100 g, increased trabecular bone mass by 24-36%, respectively, compared to the 43-61% increase induced by PTH at 8 micrograms/100 g. Unlike PTH, which increased cortical bone mass by 15-25%, PTHrP increased cortical bone mass only at the highest dose tested, 32 micrograms/100 g. Bone forming surfaces but not bone apposition rate were increased by PTH and PTHrP while resorption measures remained comparable to control values. Although serum Ca and Pi remained in the physiological range for all rats, the values for PTHrP-treated rats were consistently higher. In conclusion, PTHrP (1-34) was less potent and less effective than PTH (1-34) in inducing an anabolic response in bone in vivo.     

Daily treatment with human recombinant parathyroid hormone-(1-34), LY333334, for 1 year increases bone mass in ovariectomized monkeys.

PTH stimulates bone formation to increase bone mass and strength in rats and humans. The aim of this study was to determine the skeletal effects of recombinant human PTH-(1-34) [rhPTH-(1-34)] in monkeys, as monkey bone remodeling and structure are similar to those in human bone. Adult female cynomolgus monkeys were divided into sham-vehicle (n = 21), ovariectomized (OVX)-vehicle (n = 20), and OVX groups given daily s.c. injections of rhPTH-(1-34) at 1 (n = 39) or 5 (n = 41) microg/kg for 12 months. Whole body bone mineral content was measured, as was bone mineral density (BMD) in the spine, proximal tibia, midshaft radius, and distal radius. Serum and urine samples were also analyzed. rhPTH-(1-34) treatment did not influence serum ionized Ca levels or urinary Ca excretion, but depressed endogenous PTH while increasing serum calcitriol levels. Compared to that in the OVX group, the higher dose of rhPTH-(1-34) increased spine BMD by 14.3%, whole body bone mineral content by 8.6%, and proximal tibia BMD by 10.8%. Subregion analyses suggested that the anabolic effect of rhPTH-(1-34) on the proximal tibia was primarily in cancellous bone. Similar, but less dramatic, effects on BMD were observed with the lower dose of rhPTH-(1-34). Daily s.c. rhPTH-(1-34) treatment for 1 yr increases BMD in ovariectomized monkeys without inducing sustained hypercalcemia or hypercalciuria.     

Restoration of bone mass in the severely osteopenic senescent rat

Studies in humans and rats suggest that age impairs the ability to form bone. This impairment may be due to a depletion or deficit in osteoprogenitor stem cells. Such a deficit would be expected to reduce the ability of the skeleton to respond to therapy designed to restore lost bone. This study evaluated whether severely osteopenic senescent rats are capable of responding to a potent anabolic factor in bone, prostaglandin E2 (PGE). Growing female Sprague Dawley rats were ovariectomized at 3 months and aged until the start of treatment at 23 months. Rats were treated daily with PGE (3 mg/kg sc) or vehicle for 56 days. Tibiae were harvested for bone histomorphometry and femora were obtained for mRNA analysis of bone matrix proteins. The cancellous bone area was fivefold greater in PGE-treated rats than in vehicle-treated controls and not different from age-matched ovary-intact rats. PGE approximately doubled the bone-forming surface and the mineral apposition rate and increased the bone formation rate fourfold. The increased cancellous bone area in PGE-treated rats was primarily due to an increase in osteoblasts over osteoclasts. One hundred percent of the endocortical surface and 72 +/- 9% of the periosteal surface of cortical bone was undergoing mineralization in PGE-treated rats, whereas no mineratization was evident in vehicle-treated rats. An architectural analysis of cancellous bone indicates that trabecular number and thickness were increased and separation decreased in the treated rats. Imaging by microcomputed tomography further revealed that with PGE treatment, trabeculae in the medial plane of the proximal tibial metaphysis were more robust and continuous with the endocortical surface. PGE also significantly induced message levels for the prepro-alpha (I) subunit of type I collagen (collagen), osteonectin, and osteocalcin. In summary, bone mass can be restored to severely osteopenic senescent rats, suggesting that aging does not necessarily diminish the capacity of the skeleton to form bone.     

Follicle-stimulating hormone increases bone mass in female mice

Elevated follicle-stimulating hormone (FSH) activity is proposed to directly cause bone loss independent of estradiol deficiency in aging women. Using transgenic female mice expressing human FSH (TgFSH), we now reveal that TgFSH dose-dependently increased bone mass, markedly elevating tibial and vertebral trabecular bone volume. Furthermore, TgFSH stimulated a striking accrual of bone mass in hypogonadal mice lacking endogenous FSH and luteinizing hormone (LH) function, showing that FSH-induced bone mass occurred independently of background LH or estradiol levels. Higher TgFSH levels increased osteoblast surfaces in trabecular bone and stimulated de novo bone formation, filling marrow spaces with woven rather than lamellar bone, reflective of a strong anabolic stimulus. Trabecular bone volume correlated positively with ovarian-derived serum inhibin A or testosterone levels in TgFSH mice, and ovariectomy abolished TgFSH-induced bone formation, proving that FSH effects on bone require an ovary-dependent pathway. No detectable FSH receptor mRNA in mouse bone or cultured osteoblasts or osteoclasts indicated that FSH did not directly stimulate bone. Therefore, contrary to proposed FSH-induced bone loss, our findings demonstrate that FSH has dose-dependent anabolic effects on bone via an ovary-dependent mechanism, which is independent of LH activity, and does not involve direct FSH actions on bone cells.     

Abnormal bone architecture and biomechanical properties with near-lifetime treatment of rats with PTH

Skeletal effects are described for near-lifetime treatment of young, female rats with recombinant human PTH (1-34) (PTH). Rats (5-8 wk of age) were administered 0, 5, 30, or 75 microg/kg x d sc PTH for up to 2 yr, as part of an oncogenicity evaluation, which is required by regulatory agencies for potential chronic therapies. Proliferative lesions were observed in the skeleton as described in Vahle et al. (1 ); in this paper, we describe the quantitative bone data for this study. In the appendicular skeleton, PTH stimulated trabecular and endocortical mineral apposition to the near exclusion of marrow spaces at 5 microg/kg, with some periosteal apposition at 30 microg/kg, followed by considerable periosteal apposition and altered geometry at 75 microg/kg. Increased bone mass was observed for all treatment groups that substantially exceeded normal levels attained by vehicle controls and exceeded skeletal efficacy reported previously for similar doses in shorter-term studies. Dose-dependent increases in osteocalcin levels and a linear increase in wet weight of femora were observed for the entire treatment duration, suggesting nearly continuous PTH stimulation of osteoblasts and skeletal growth throughout life. Histology showed many osteocytes and prominent osteoblasts, but a conspicuous absence of osteoclasts. Morphometry showed a lack of distinction between trabecular and cortical bone. Biomechanics of vehicle controls showed that optimal mechanical integrity for the normal skeleton is observed at about 11 months of age. PTH greatly strengthened and stiffened vertebra and femora; however, the midshaft showed reduced toughness and increased brittleness with treatment, which was not the case for vertebra. Related studies of 6 and 9 months duration showed that the optimal duration for PTH skeletal efficacy was about 6 months in rats, based on toughness, strength, ultimate displacement, and architecture, especially for cortical bone. Therefore, treatment duration is an under appreciated aspect of PTH pharmacology; and PTH skeletal effects are a complex function of dose and duration. Comparative analyses showed that short-term treatment (6 months or less) is more advantageous than near-lifetime treatment, because PTH stimulates skeletal growth throughout life, resulting in abnormal architecture and untoward biomechanical properties in rats.