去卵巢大鼠骨髓源性破骨样细胞增殖、分化的改变及雌激素的影响

目的　通过体外骨髓细胞培养诱导破骨样细胞 (osteoclast likecells ,OLC)的形成 ,观察去卵巢对成年大鼠OLC形成及活性的影响以及给予雌激素后的改变。　方法　 3月龄SD大鼠分为对照组、去卵巢组及雌激素替代组。术后 12周处死大鼠 ,取股骨分离骨髓细胞 ,在条件培养液中诱导其向破骨细胞分化。活体观察OLC形成情况并于培养的第 6天行细胞染色 ,以抗酒石酸酸性磷酸酶(TRAP)染色 (+)、细胞核≥ 3个的细胞为OLC ,计数各组OLC及骨陷窝。　结果　 3组中去卵巢组OLC出现早且数量〔(2 7 75± 0 92 )个 /玻片〕明显高于其它两组〔(17 93± 0 6 9)个 /玻片和 (12 81±0 6 1)个 /玻片 ,P <0 0 1〕。雌激素处理能明显抑制OLC的形成 ,但雌激素替代组的OLC仍多于对照组 (P <0 0 5 )。骨陷窝形成的变化与OLC数量改变一致。　结论　本实验结果显示大鼠去卵巢后 ,骨髓干细胞分化形成OLC数量明显增多 ,且活性增强。补充雌激素能够有效抑制OLC形成增加及其活性增强。故雌激素抑制骨吸收的机制至少部分是作用于骨髓干细胞向破骨细胞的分化。     

Effects of dietary sodium chloride loading on parathyroid function, 1,25-dihydroxyvitamin D, calcium balance, and bone metabolism in female rats during chronic prednisolone administration

The effects of dietary sodium chloride supplements (8 g/100 g diet) on parathyroid function, serum 1,25-dihydroxyvitamin D [1,25-(OH)2D], calcium balance, bone metabolism, and bone composition were studied in rats treated with prednisolone (2 mg/kg w X day) for 12 weeks. Animals on a low calcium diet (0.1% Ca) received the following treatments: group 1, control; group 2, NaCl; group 3, prednisolone; group 4, NaCl plus prednisolone. Parathyroid function was assessed indirectly from urinary cAMP excretion: bone resorption was estimated by studying urinary hydroxyproline excretion and mobilization of 45Ca from bone. Dietary salt loading increased the urinary excretion of calcium, 45Ca, cAMP, and hydroxyproline and raised serum 1,25-(OH)2D and net calcium absorption, but lowered calcium retention, femoral calcium, and total body calcium. Prednisolone slowed body growth and lowered net calcium absorption, calcium retention, femoral calcium, and total body calcium. Urinary calcium excretion was higher in rats receiving salt and prednisolone in combination than in animals taking salt without prednisolone, but other responses to salt and prednisolone were independent. Thus, salt and prednisolone each elicit osteopenia, and salt causes bone loss in rats receiving prednisolone. The osteopenic effect of salt is attributed to primary augmentation of urinary calcium excretion and secondary increases in PTH-medicated bone resorption. Although salt-treated rats have higher blood levels of 1,25-(OH)2D, bone loss occurs because alimentary calcium absorption is not elevated sufficiently to offset urinary calcium losses. Prednisolone lowers bone formation and net calcium absorption without lowering serum 1,25-(OH)2D values. The parathyroid-vitamin D axis remains intact in prednisolone-treated rats, as they show increases in PTH and 1,25-(OH)2D after salt treatment.     

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.     

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.     

仙灵骨葆对去势大鼠骨量和生物力学性能的影响

目的探讨仙灵骨葆对骨质疏松(OP)大鼠骨量、骨代谢和生物力学性能的影响。方法 3月龄雌性SD大鼠24只分为3组,每组8只:正常对照组(N)、卵巢切除组(OVX)、卵巢切除+仙灵骨葆治疗组(XLGB)。除N组外,其余两组行卵巢切除术,6 w后XLGB组给予药物干预:250 mg.kg-1.d-1,OVX组给予等量生理盐水,8 w后处死所有大鼠。留取尿液、血清检测血PINP值、尿DPYD/Cr、NTX/Cr值。取左侧股骨行骨密度测定,取左侧胫骨制备硬组织不脱钙切片,备行骨组织形态计量学检测,取右侧股骨行三点弯曲试验,检测其最大载荷。结果 OVX组血PINP、尿DPYD/Cr、尿NTX/Cr值显著高于N组,XLGB能显著降低血PINP、尿DPYD/Cr、尿NTX/Cr值,但仍显著高于N组。OVX组股骨全长及近、中、远三段骨密度均显著低于N组,XLGB组近、远端骨密度显著高于OVX组。BV/TV在OVX组显著低于N组,XLGB组显著高于OVX组;OVX、XLGB组骨吸收指标Oc.N、Er.Pm均显著高于N组,XLGB组Oc.N、Er.Pm显著低于OVX组,BFR/BV显著高于OVX组。最大载荷三组之间无显著差别。结论仙灵骨葆灌胃可抑制卵巢切除大鼠骨量丢失,其机制与促进骨形成、抑制骨吸收,降低骨转换水平,进而维持骨量及微观结构有关。     

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.     

Calcitonin versus Clodronate in the Prevention of Ovariectomy-Induced Osteopenia in Rats

The effects of salmon calcitonin and clodronate were compared in ovariectomised rats. Sixty female Wistar rats (∼260 g in weight) were fed the same diet and had the same living conditions. The rats were divided into the following groups: 15 rats with sham ovariectomy and no drug treatment (Sham-OVX); 45 rats with bilateral ovariectomy subdivided into 15 rats not receiving drug treatment (OVX group), 15 rats treated with subcutaneous salmon calcitonin, 2 U/kg/day every 2 days (OVX + CT group) and 15 rats treated with subcutaneous clodronate, 5 mg/kg/day every 2 days (OVX + Cl group). Sixty days after surgery, the rats were sacrificed and their femurs and fifth lumbar vertebrae were dissected and cleaned of soft tissue. Femur length, vertebral height, and bone mineral content and bone mineral density of the femur and fifth lumbar vertebra by dual-energy X-ray absorptiometry were measured. Calcitonin had a significant and stronger effect in preventing ovariectomy-induced osteopenia in the femur (OVX + CT vs OVX groups, p<0.0001); both calcitonin and clodronate had a significant effect on the fifth lumbar vertebra, which was greater in the calcitonin group (OVX + CT vs OVX + Cl groups, p<0.005). These findings indicate that calcitonin has a protective effect on both the axial (trabecular bone) and peripheral (cortical bone) skeletons, but clodronate only has a protective effect on the axial skeleton. Received: 28 May 1999 / Accepted: 29 July 1999     

雌激素对大鼠去卵巢后白细胞介素-6介导的骨吸收的影响

目的观察雌激素对去卵巢大鼠骨髓细胞白细胞介素6(IL6)、IL6受体、gp130基因表达以及骨髓源性破骨细胞形成的影响。方法健康3月龄雌性SD大鼠72只,随机平均分为假手术对照组、去卵巢组和雌激素组(苯甲酸雌二醇02mg/kg,皮下注射,每周1次)。分别于术后2、4、6、12周每组各取6只大鼠骨髓细胞作细胞培养和提取RNA。培养第6天计数破骨细胞数,第12周取左侧胫骨作骨形态计量学检测。结果骨形态学显示去卵巢后出现骨吸收亢进,雌激素对其有抑制作用。去卵巢后2周,去卵巢组破骨细胞形成数即多于对照组(1450±169对901±141,P<005),骨髓细胞IL6和IL6受体mRNA表达均显著升高(P<005,P<001),第4～6周,上述改变达高峰,至第12周仍呈有意义增高;从2～12周,上述各指标雌激素组均明显低于去卵巢组(P<005,P<001)。各组未见gp130基因表达水平有明显变化。结论雌激素可以抑制大鼠去卵巢后骨髓源性破骨细胞的生成,这一效应可能与其抑制骨髓细胞在去卵巢后过度表达IL6、IL6受体基因有关。     

Amylin inhibits ovariectomy-induced bone loss in rats

Amylin (AMY), a peptide co-secreted with insulin by pancreatic beta-cells, inhibits bone resorption and stimulates osteoblastic activity. The ovariectomized (OVX) rat is an established animal model for human osteoporosis. Thus, the present experiment was performed to study the effects of AMY on estrogen deficiency-induced bone loss in rats. Thirty-one 6-month-old Wistar rats were randomized by body weight (BW) into two groups. The first underwent surgical OVX (n=21). The second was sham-operated (SH; n=10). Sixty days after surgery, 11 OVX rats were s.c. injected with rat AMY (3 microg/100 g BW/day, for 30 days; OVX+AMY), and 10 with solvent alone in the same way (0.15 ml/100 g BW; OVX). Each rat, housed in an individual cage, was fed daily the mean quantity of diet consumed the day before by SH rats. This diet contained 0.24% calcium and 0. 16% phosphorus. The 31 animals were killed on day 90. No difference in daily weight gain and BW was observed between groups. Neither AMY treatment nor OVX had any significant effect upon femoral morphology, femoral failure load, diaphyseal femoral density (representative of cortical bone) and total femoral calcium content. Nevertheless, both distal metaphyseal (representative of cancellous bone) and total femoral bone densities were higher in SH and OVX+AMY than in OVX rats. The highest plasma osteocalcin concentration was measured in OVX+AMY rats. Simultaneously, urinary deoxypyridinoline excretion was lower in OVX+AMY than in OVX rats. These results indicate that in OVX rats, AMY treatment inhibited trabecular bone loss both by inhibiting resorption and by stimulating osteoblastic activity.     

葛根素对去卵巢大鼠机体骨代谢影响的观察

目的 观察葛根素对去卵巢大鼠机体骨代谢的影响,探讨其对雌激素缺乏引起的骨质疏松症的治疗作用.方法 3月龄清洁级SD大鼠60只,背驮式切除双侧卵巢后每日灌胃葛根素5 mg/kg(P-5组),10 mg/kg(P-10组)和20 mg/kg(P-20组),并设假手术组(Sham),模型组(OVX)和己烯雌酚阳性对照组(E).3个月后处死动物,测定大鼠胫骨干重、灰分重量和矿物质含量,胫骨Ca、P含量以及血清相关骨代谢指标.结果 与OVX组相比,葛根素各组的胫骨矿物质含量(mg/g)均有增加(574±17,590±22和597±18),其中P-20组差异显著(P＜0.05);葛根素各组的胫骨Ca含量(mg/g)高于OVX组 (132±10,222±7,228±8),其中P-10,P-20两组差异显著(P＜0.05,P＜0.01),说明服用葛根素后大鼠骨量得到增加;同时,葛根素各组的碱性磷酸酶(U/L)与OVX组有所降低(101±26,90±20,71±15),其中P-10,P-20两组差异显著(P＜0.05,P＜0.01),说明去卵巢大鼠骨的高转换状态得到改善.结论 葛根素能抑制去卵巢大鼠骨量的丢失,对骨代谢有较好的调节作用,对雌激素缺乏引起的骨质疏松症有一定的治疗作用.     

雌激素对去卵巢大鼠内脏脂肪细胞脂肪因子表达水平的影响

目的 观察雌激素对去卵巢大鼠内脏脂肪细胞瘦素、脂联素、抵抗素和肿瘤坏死因子-α(TNF-α)表达水平的影响,探讨雌激素对体脂分布的影响机制.方法 6周龄Sprauge-Dawley雌性大鼠30只,采用随机数字表法分成3组:假手术组、去卵巢组和去卵巢+戊酸雌二醇组(OVX+E2组),每组10只.术后1周,OVX+E2组大鼠每天按1 mg/kg体重灌胃戊酸雌二醇水溶液,其他组大鼠灌胃等体积蒸馏水.连续给药12周后,腹主动脉取血,迅速剥离内脏脂肪组织.采用全自动生化分析仪检测血脂、血糖.采用免疫组化染色、实时荧光定量RT-PCR和Western印迹检测脂肪细胞瘦素、脂联素、抵抗素和TNF-α的表达.结果 3组血清瘦素、脂联素和抵抗素水平差异无统计学意义(P均＞0.05),但去卵巢组TNF-α水平显著高于假手术组(F=4.785,P＜0.05).免疫组化显示,与假手术组相比,去卵巢组内脏脂肪组织中瘦素表达明显减弱,而脂联素、抵抗素和TNF-α表达明显增强;与去卵巢组相比,OVX+E2组内脏脂肪组织中瘦素表达明显增强,脂联素、抵抗素和TNF-α表达明显减弱(F =3.712 ～5.198,P均＜0.05).3组内脏脂肪细胞瘦素mRNA和蛋白表达水平差异无统计学意义(P均＞0.05);去卵巢组内脏脂肪细胞脂联素、抵抗素和TNF-α的mRNA和蛋白表达水平显著高于假手术组,而OVX+E2组内脏脂肪细胞脂联素、抵抗素和TNF-α的mRNA和蛋白表达水平显著低于去卵巢组(F=3.175～5.342,P均＜0.05).结论 雌激素可通过下调去卵巢大鼠内脏脂肪细胞脂联素、抵抗素和TNF-α的表达,进而影响去卵巢大鼠体脂再分布.     

Separate and combined effects of growth hormone and parathyroid hormone on cortical bone osteopenia in ovariectomized aged rats.

The focus of this study is on whether cortical osteopenia occurs in ovariectomized aged female rats, and if so, whether growth hormone (GH) and parathyroid hormone (PTH) independently or together (GH+PTH) can rebuild the lost cortical bone. Tibio-fibula junction was analyzed by histomorphometry and peripheral quantitative computerized tomography (pQCT) densitometry. Significant loss of cortical bone area (Ct. BAr), cortical bone mineral content (Ct. BMC), cortical thickness (Ct. Th) and increase of endocortical perimeter occurred 4 months after ovariectomy. The rats were given GH, PTH, GH+PTH or vehicle for 2 months and sacrificed. GH, PTH and GH+PTH increased Ct. BAr, Ct. BMC, Ct. Th, periosteal perimeter, periosteal double-labeled perimeter, mineral apposition rate, and bone formation rate, but decreased marrow area. PTH and GH+PTH decreased endocortical perimeter, and increased endocortical double labeled perimeter and bone formation rate. In conclusion, ovariectomy induced cortical bone loss in aged rats by increasing endocortical bone resorption. Growth hormone increased periosteal bone formation, while PTH stimulated endocortical bone formation and in combination GH+PTH produced complementary effects thereby reversing osteopenia.     

Direct effects of 17 beta-estradiol on trabecular bone in ovariectomized rats.

High-affinity nuclear binding sites for 17 beta-estradiol (17 beta E2) were recently found in bone cells; however, the mechanism by which estrogen exerts its effect on bone in vivo is still unknown. To study if estrogen acts on bone directly, we used an experimental model in which test substances are infused locally into rat femur trabecular bone. Sprague-Dawley rats weighing 150-160 g were ovariectomized (OVX) and 14 days later a polyethylene tube (1 mm in diameter) connected to an Alzet osmotic minipump was implanted into the distal femur 9 mm from the joint. 17 beta E2 (24 microliters/day at 0.01-1 nM), 17 alpha-estradiol (17 alpha E2) (24 microliters/day at 1 nM), or phosphate-buffered saline (NaCl, 8 g/liter; KCl, 0.2 g/liter; KH2PO4, 0.2 g/liter; Na2HPO4.7H2O, 2.16 g/liter) was infused for 8 days. The contralateral limb remained intact. Animals were sacrificed and bones were examined by histomorphometry. Ovariectomy caused a 50% loss in trabecular bone volume (TBV) in the secondary spongiosa (from 20.3% +/- 1.7% to 9.6% +/- 1.1%; mean +/- SEM), a 2-fold increase in osteoclast number (to 4.0 +/- 0.4 per mm), a 3-fold increase in relative resorption surfaces (to 24.8% +/- 2.9%), a 9-fold increase in osteoblast number (to 11.3 +/- 2.1 per mm), and an 8-fold increase in relative osteoid surface (to 9.6% +/- 1.7%). The local infusion of 17 beta E2 for 8 days into OVX rats (i) restored the TBV dose dependently to 75% and 85% of control (non-OVX) levels, at 0.1 nM and 1 nM 17 beta E2, respectively; (ii) decreased osteoclast number and the relative resorption surface to control (non-OVX) levels; and (iii) further increased osteoblast number and the relative osteoid surface dose dependently (by 5-fold at 1 nM 17 beta E2). Phosphate-buffered saline infusion was without effect. Infusion of 17 alpha E2 had no effect on TBV, osteoclast number, or resorption surface but increased slightly the osteoblast number and the osteoid surface. Its potency was 1/100 that of 17 beta E2. The local infusion of 17 beta E2 or 17 alpha E2 had no effect on body or uterine weight. We conclude from these findings that estrogen delivered directly to the bone of OVX rats in vivo at 2.4 and 24 fmol/day acted locally to inhibit bone resorption and stimulate bone formation.     

Skeletal effects of calcitonin in ovariectomized rats

Although calcitonin (CT) has been shown to be effective for the prevention of bone loss in early postmenopausal women, the skeletal effects of the hormone specifically during the early stages of estrogen deficiency have not been characterized histomorphometrically to date. The current study involves use of the ovariectomized (OVX) rat as an animal model for early postmenopausal bone loss to perform such a histomorphometric analysis. One group of OVX rats was injected sc with salmon CT on alternate days for a 6-week period. Additional groups of OVX and sham-operated control rats were treated with vehicle alone. In comparison to control rats, the proximal tibia of vehicle-treated OVX rats were characterized by a 3-fold decrease in cancellous bone volume and significant increases in osteoblast surface (+200%), osteoclast surface (+143%), mineralizing surface (+111%), mineral apposition rate (+36%), bone formation rate (+181%), and longitudinal bone growth (+38%). In contrast, treatment of OVX rats with CT normalized tibial cancellous bone volume and significantly decreased all of the above cellular- and fluorochrome-based indices of bone turnover to near control levels. The results indicate that CT treatment depresses bone turnover and prevents the development of osteopenia in OVX rats. These findings are consistent with the bone protective effect of CT in early postmenopausal women and further support the OVX rat as an animal model for the preclinical evaluation of prophylactic treatments for postmenopausal bone loss.     

Combined intervention of soy isoflavone and moderate exercise prevents body fat elevation and bone loss in ovariectomized mice

Body fat accumulation and bone loss are both often associated with estrogen deficiency following menopause. In this study, we examined whether soy isoflavone, one of the phytoestrogens, and moderate exercise interventions exhibit cooperative effects on body composition and bone mass in ovariectomized (OVX) mice. Eight-week-old female mice were assigned to 6 groups: (1) sham-operated (sham); (2) OVX; (3) OVX with received a soy isoflavone diet (OVX+ISO); (4) OVX with exercised on a treadmill (OVX+EX); (5) OVX with given both isoflavone and exercise (OVX+ISO&EX ); and (6) OVX with treated with 17 beta-estradiol subcutaneously (OVX+E2). Body composition and bone mineral density (BMD) were estimated by dual-energy x-ray absorptiometry (DXA). After the 6-week intervention, whole body fat (%) in the OVX group showed significantly higher than that in the sham group. Intervention of exercise and isoflavone alone partially inhibited OVX-induced body fat gain, and the combined intervention as well as E2 treatment completely restored fat mass to the sham level. Lean body mass in the whole body was not different in OVX group compared with that in OVX+ISO, OVX+EX, and OVX+E2 groups, but it was significantly higher in OVX+ISO&EX than in other groups. BMD of the whole body, lumbar spine, or femur showed significantly reduced by OVX, and the bone loss was partially inhibited by intervention of exercise or isoflavone alone. However, the combined intervention completely restored the bone mass to the level of sham, as did E2. Serum total cholesterol was significantly increased by OVX, which was normalized by the combined intervention or E2 treatment. These results demonstrate that combined intervention of soybean isoflavone and exercise prevented body fat accumulation in the whole body with an increase in lean body mass and restoration of bone mass, and reduced high serum cholesterol in OVX mice.     

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.     

Increased bone formation and bone mass induced by sclerostin antibody is not affected by pretreatment or cotreatment with alendronate in osteopenic, ovariectomized rats

Clinical studies have revealed a blunting of the bone anabolic effects of parathyroid hormone treatment in osteoporotic patients in the setting of pre- or cotreatment with the antiresorptive agent alendronate (ALN). Sclerostin monoclonal antibody (Scl-Ab) is currently under clinical investigation as a new potential anabolic therapy for postmenopausal osteoporosis. The purpose of these experiments was to examine the influence of pretreatment or cotreatment with ALN on the bone anabolic actions of Scl-Ab in ovariectomized (OVX) rats. Ten-month-old osteopenic OVX rats were treated with ALN or vehicle for 6 wk, before the start of Scl-Ab treatment. ALN-pretreated OVX rats were switched to Scl-Ab alone or to a combination of ALN and Scl-Ab for another 6 wk. Vehicle-pretreated OVX rats were switched to Scl-Ab or continued on vehicle to serve as controls. Scl-Ab treatment increased areal bone mineral density, volumetric bone mineral density, trabecular and cortical bone mass, and bone strength similarly in OVX rats pretreated with ALN or vehicle. Serum osteocalcin and bone formation rate on trabecular, endocortical, and periosteal surfaces responded similarly to Scl-Ab in ALN or vehicle-pretreated OVX rats. Furthermore, cotreatment with ALN did not have significant effects on the increased bone formation, bone mass, and bone strength induced by Scl-Ab in the OVX rats that were pretreated with ALN. These results indicate that the increases in bone formation, bone mass, and bone strength with Scl-Ab treatment were not affected by pre- or cotreatment with ALN in OVX rats with established osteopenia.     

Protective effect of an aldose reductase inhibitor against bone loss in galactose-fed rats: possible involvement of the polyol pathway in bone metabolism.

Many patients with diabetes mellitus show a moderate reduction in bone mass. Our recent in vitro studies showed that sustained exposure of osteoblast-like MG-63 cells to high glucose by itself impairs their functions partly via the polyol pathway. To investigate the role of hyperglycemia in the etiology of diabetic osteopenia in vivo separately from insulin deficiency, we determined whether epalrestat, an aldose reductase (AR) inhibitor (ARI), lessens the abnormalities in calcium (Ca) metabolism in galactose-fed rats. Weight gain was impaired in the rats, which was not altered by epalrestat. Galactose feeding temporarily enhanced bone resorption as reflected by increased biochemical markers for bone resorption (urinary excretion of pyridinoline [PYR] and deoxypyridinoline [DPYR]) at 1 to 3 months, which were significantly decreased by epalrestat. Epalrestat also restored the positive correlation between a bone-formation marker (serum osteocalcin [OC]) and a bone-resorption marker (urinary DPYR excretion) at 6.5 months. Histomorphometric analysis of bone performed 6.5 months after galactose feeding showed that both the bone volume and osteoblast numbers in the tibia, which were significantly suppressed by galactose feeding, were partly restored to a significant extent by the simultaneous administration of epalrestat. In summary, epalrestat partially protected against the development of osteoblast dysfunction and reduced the temporary increase in biochemical markers for bone resorption induced by galactose feeding, with a resultant increase in bone volume, suggesting that the polyol pathway may be intimately involved in the development of abnormal bone metabolism in galactose-fed rats.     

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.     

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.