Bisphosphonate administration alters subcellular localization of vacuolar-type H(+)-ATPase and cathepsin K in osteoclasts during experimental movement of rat molars

This study was designed to clarify the effects of bisphosphonate (BP) administration on structure and functions of osteoclasts in alveolar bone resorption during experimental movement of rat molars. To produce orthodontic force, elastic band was inserted between the upper first and second molars for 4 days, and dissected maxillae were then examined by means of light and electron microscopic immunocytochemistry for vacuolar-type H(+)-ATPase and lysosomal cystein proteinase, cathepsin K in osteoclasts. Vacuolar-type H(+)-ATPase and cathepsin K in osteoclasts are the most important enzymes for demineralization of apatite crystals and degradation of bone type-I collagen, respectively. At 1 day before elastic band insertion, BP was administered intraperitoneally. Control rats received the same volume of physiologic saline. In BP-administered rats, most osteoclasts exhibited either irregularly-formed ruffled borders and clear zones or only clear zones of various degrees of extension. Subcellular localization and expression of both vacuolar-type H(+)-ATPase and cathepsin K was significantly decreased in such osteoclasts with impaired ruffled borders and/or only clear zones by BP administration. In particular, cathepsin K secretion by osteoclasts towards resorption lacunae was markedly inhibited by BP administration. Our results indicate for the first time that BP administration significantly impair the osteoclast structure and reduces expression of both vacuolar-type H(+)-ATPase and cathepsin K in osteoclasts during tooth movement.     

Effect of high molecular weight water-soluble chitosan on the trabecular bone and thickness in ovariectomized rats

High molecular weight water-soluble chitosan(WSC), having an average molecular weight of 300,000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the trabecular bone area and thickness in ovariectomized(OVX) rats decreased by almost 50% from those in sham-operated rats. WSC was evaluated for inhibition of the progress of bone loss induced by OVX rats. We measured bone histomorphometry in sham, OVX or WSC-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham-operated rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of WSC at a concentration of 15 mg/kg/daily for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of WSC for 7 weeks. In OVX rats, free T(3) was normal in all cases, whereas free T(4) was significantly increased. Although there was no difference between OVX and WSC-administered rats in T(3) level, we have found significant difference between them in T(4) level. These results strongly suggest that WSC is effective in preventing the development of bone loss induced by OVX in rats.     

Osteointegration of bioactive glass-coated and uncoated zirconia in osteopenic bone: an in vivo experimental study

In elderly and osteoporotic patients an age-related loss of osteoinductivity could be the biological cause of implant failure regardless of the high quality of the implanted device. yttria stabilized tetragonal zirconia (YSTZ), either coated with the bioactive glass named RKKP bioglaze (RKKP) or uncoated, was implanted in the distal femurs of sham-operated and ovariectomized female rats. Animals were sacrificed at 30 and 60 days. Histomorphometry and microhardness tests were performed to assess osteointegration rate as well as bone quality around the implants. Significant decreases (p < 0.0005) in trabecular bone volume, BV/TV (41%), trabecular bone surface BS/TV (33%), trabecular thickness Tb.Th (20%), and trabecular number Tb.N (32%), together with a significant increase in trabecular separation Tb.Sp (184%), were found for the osteopenic rats compared with the sham-operated rats. At both experimental times the RKKP coating ensured a better osteointegration rate with higher AI values than the uncoated YSTZ, even when osteopenic rats were used (48% at 30 days and 12% at 60 days). No differences were observed at the bone-biomaterial interfaces for either material when comparing sham-operated with osteopenic rats. The present results demonstrate that the RKKP bioactive glass used as a coating ensures a high osteointegration rate even in osteoporotic bone, which is already visible from postoperative day 30 and is still apparent on day 60.     

Bone wound healing after maxillary molar extraction in ovariectomized aged rats: quantitative backscattered electron image analysis

The processes of bone wound healing after maxillary molar extraction in ovariectomized aged rats were examined by means of quantitative backscattered electron image analysis and energy-dispersive X-ray microanalysis. Six-month-old female rats were either sham-operated or underwent bilateral ovariectomy (OVX), and 60 days postoperatively, the maxillary first molars were extracted. On post-extraction days 7, 30, and 60, the dissected and resin-embedded maxillae were micromilled in the transverse direction through the extracted alveolar sockets, and new bone formation on the buccal maxillary bone surface and within the extracted alveolar sockets was examined. In both sham-operated control and OVX rats, new bone formation was recognized on the buccal bone surface, as well as within the extracted sockets, and increased daily through to day 60. In comparison to sham-operated controls, new bone formation in OVX rats was significantly decreased both on the buccal bone surface and within the extracted sockets. Our results suggest that bone wound healing by new bone formation after maxillary molar extraction is significantly decreased in OVX-induced osteoporosis.     

Effect of High Molecular Weight Water-Soluble Chitosan on the Trabecular Bone and Thickness in Ovariectomized Rats

High molecular weight water-soluble chitosan(WSC), having an average molecular weight of 300,000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the trabecular bone area and thickness in ovariectomized(OVX) rats decreased by almost 50% from those in sham-operated rats. WSC was evaluated for inhibition of the progress of bone loss induced by OVX rats. We measured bone histomorphometry in sham, OVX or WSC-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham-operated rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of WSC at a concentration of 15 mg/kg/daily for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of WSC for 7 weeks. In OVX rats, free T₃ was normal in all cases, whereas free T₄ was significantly increased. Although there was no difference between OVX and WSC-administered rats in T₃ level, we have found significant difference between them in T₄ level. These results strongly suggest that WSC is effective in preventing the development of bone loss induced by OVX in rats.     

Effect of High Molecular Weight Water-Soluble Chitosan on the Trabecular Bone and Thickness in Ovariectomized Rats

High molecular weight water-soluble chitosan(WSC), having an average molecular weight of 300,000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the trabecular bone area and thickness in ovariectomized(OVX) rats decreased by almost 50% from those in sham-operated rats. WSC was evaluated for inhibition of the progress of bone loss induced by OVX rats. We measured bone histomorphometry in sham, OVX or WSC-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham-operated rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of WSC at a concentration of 15 mg/kg/daily for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of WSC for 7 weeks. In OVX rats, free T₃ was normal in all cases, whereas free T₄ was significantly increased. Although there was no difference between OVX and WSC-administered rats in T₃ level, we have found significant difference between them in T₄ level. These results strongly suggest that WSC is effective in preventing the development of bone loss induced by OVX in rats.     

绝经后骨质疏松症模型体视学测量和机理初探

目的:观测去势术后大鼠骨组织体视学的改变,复制绝经后骨质疏松症的动物模型,初步探讨骨质疏松症的发病机制。方法:将31只3月龄雌性SD大鼠随机分为卵巢切除术组(OVX)和假手术组(sham),术后28d和56d分别处死。测量子宫湿重,骨矿物密度(BMD)和骨组织形态计量参数,分析骨组织微结构的变化。结果:OVX组术后28d和56d大鼠子宫湿重和股骨远端1/3处骨密度均显著少于sham组(P＜0.05);OVX组股骨远端和胫骨近端的干骺端骨小梁面积百分率显著少于sham组(P＜0.01)。结论:卵巢切除术后大鼠股骨远端骨矿物密度和骨组织形态计量学参数稳步下降,胫骨近端骨矿物密度和骨组织形态计量学参数迅速下降而且不稳定;雌激素减少导致的骨质疏松主要发生在长骨的干骺端,骨骺受影响较少;骨组织形态计量参数中骨小梁面积百分比敏感性和稳定性较高。     

Comparative effects of milk and soymilk on bone loss in adult ovariectomized osteoporosis rat

The present study was designed to investigate the comparative effects of milk and soymilk on the bone morphology of ovariectomized rat model with postmenopausal osteoporosis. Fourty 12-week-old female Spraque-Dawley rats were randomly divided into the following 5 groups: intact control (control), sham-operated (sham), ovariectomized (OVX), ovariectomized and fed milk (milk), ovariectomized and fed soymilk (soymilk) groups. Rats of OVX, milk and soymilk groups were bilaterally ovariectomized. Milk or soymilk of 3 ml was administered by gavage via an intragastric tube per day for 6 weeks. The distal femoral metaphysis was studied morphologically, together with bone mineral density (BMD), serum parathyroid hormone (PTH) and estradiol levels. In the OVX group, the trabecular bone volume was significantly reduced. The percentage area of resorbing surface was high. In the soymilk group, the trabecular bone volume was 68% higher and the percentage area of resorbing surface was 18% lower as compared with the OVX group. The serum estradiol level rose 48% and the serum PTH level fell 26% in the soymilk group. In the milk group, the trabecular bone volume had the tendency to be high, but there was no significant change of the bone morphology. The present study provides certain evidence that the soymilk has beneficial effects in preventing bone loss in rats induced by ovarian hormone deficiency.     

Bisphosphonate administration alters subcellular localization of vacuolar‐type H+‐ATPase and cathepsin K in osteoclasts during experimental movement of rat molars

This study was designed to clarify the effects of bisphosphonate (BP) administration on structure and functions of osteoclasts in alveolar bone resorption during experimental movement of rat molars. To produce orthodontic force, elastic band was inserted between the upper first and second molars for 4 days, and dissected maxillae were then examined by means of light and electron microscopic immunocytochemistry for vacuolar‐type H⁺‐ATPase and lysosomal cystein proteinase, cathepsin K in osteoclasts. Vacuolar‐type H⁺‐ATPase and cathepsin K in osteoclasts are the most important enzymes for demineralization of apatite crystals and degradation of bone type‐I collagen, respectively. At 1 day before elastic band insertion, BP was administered intraperitoneally. Control rats received the same volume of physiologic saline. In BP‐administered rats, most osteoclasts exhibited either irregularly‐formed ruffled borders and clear zones or only clear zones of various degrees of extension. Subcellular localization and expression of both vacuolar‐type H⁺‐ATPase and cathepsin K was significantly decreased in such osteoclasts with impaired ruffled borders and/or only clear zones by BP administration. In particular, cathepsin K secretion by osteoclasts towards resorption lacunae was markedly inhibited by BP administration. Our results indicate for the first time that BP administration significantly impair the osteoclast structure and reduces expression of both vacuolar‐type H⁺‐ATPase and cathepsin K in osteoclasts during tooth movement. Anat Rec 260:72–80, 2000. © 2000 Wiley‐Liss, Inc.     

Placenta hominis protects osteoporosis in ovariectomized rats

In China, Japan, and Korea, placenta hominis extracts (PHEs) are used clinically for the treatment of osteoporosis. The anti-osteoporotic effect of PHEs was studied. The trabecular bone area and thickness in OVX rats decreased by 50% from those in sham-operated rats; these decreases were completely inhibited by administration of PHEs for 7 weeks. Osteoclast numbers and the osteoblast surface were enhanced in OVX rats, but PHEs had no effect on these phenomena. Serum phosphorus and alkaline phosphatase in OVX rats increased compared to those in sham-operated rats, but the increases were not affected by the administration of PHEs. Thyroxine (T₄) level was stimulated in OVX rats. The extracts inhibited the T₄ level in the OVX rats. These results strongly suggest that PHEs be effective in preventing the development of bone loss induced by OVX in rats.     

Effects of hPTH (1-34) and Gosha-jinki-gan on the trabecular bone microarchitecture in ovariectomized rat tibia

Although human parathyroid hormone (1-34) [hPTH (1-34)] was reported to improve osteoporotic bone loss, little is known about the anti-osteoporotic effect of the traditional Chinese medicine, Gosha-jinki-gan (GJG). The purpose of this present study was to clarify and compare the effects of hPTH (1-34) and GJG on trabecular bone microarchitecture in ovariectomized (OVX) rat tibia by using microcomputed tomography (micro-CT). Thirty 12-week-old Sprague-Dawley female rats were underwent ovariectomy (OVX) or sham operation. Four weeks later, the ovariectomized rats were further divided into OVX, OVX + PTH, and OVX + GJG groups. hPTH (1-34) was administered subcutaneously at a dose of 20 microg/kg, 3 times/week, and OVX + GJG group received 0.05% aqueous solution of GJG as the only drinking fluid for 8 weeks respectively. The three-dimensional (3D) trabecular microarchitecture of the bone in the proximal tibial metaphysis was evaluated by micro-CT. In the OVX + PTH group, trabecular bone volume (BV/ TV), number (Tb.N) and thickness (Tb.Th) were significantly increased, structure model index (SMI) and trabecular bone pattern factor (TBPf) decreased when compared with the OVX group. In comparison to the OVX group, BV/TV and Tb.N were significantly greater, while SMI and TBPf had no marked changes in the OVX + GJG group. These results suggest that the administration of hPTH (1-34) restore the trabecular bone volume and improve the microstuctural property as well, while GJG reduce the bone loss without affecting its microstructural property in ovariectomized rats.     

Placenta Hominis Protects Osteoporosis in Ovariectomized Rats

In China, Japan, and Korea, placenta hominis extracts (PHEs) are used clinically for the treatment of osteoporosis. The anti-osteoporotic effect of PHEs was studied. The trabecular bone area and thickness in OVX rats decreased by 50% from those in sham-operated rats; these decreases were completely inhibited by administration of PHEs for 7 weeks. Osteoclast numbers and the osteoblast surface were enhanced in OVX rats, but PHEs had no effect on these phenomena. Serum phosphorus and alkaline phosphatase in OVX rats increased compared to those in sham-operated rats, but the increases were not affected by the administration of PHEs. Thyroxine (T₄) level was stimulated in OVX rats. The extracts inhibited the T₄ level in the OVX rats. These results strongly suggest that PHEs be effective in preventing the development of bone loss induced by OVX in rats.     

雌激素对骨质疏松性骨折愈合过程BMP-4基因表达的影响

目的：研究雌激素对骨质疏松性骨折愈合骨形态发生蛋白-4（bone morphogenetic protein-4,BMP-4）基因定位分布的影响。 方法： 选用健康SD大鼠，随机分为卵巢切除（OVX）组、假手术对照（sham）组和雌激素替代治疗组（OVXE）。制成胫骨骨折愈合模型并分别于骨折后不同时点处死大鼠取材。通过光镜、电镜观察形态学和超微结构变化；采用原位杂交方法观察雌激素对BMP-4基因表达影响。 结果： (1)OVX组在骨痂形态及成骨细胞和破骨细胞活性与OVXE组比较有明显差异，较sham组差异更大。（2）骨折后1-3 d BMP-4 mRNA表达强度:sham组相似文献   

Transient Effect of 17β-estradiol on Osteoporosis in Ovariectomized Rats Accompanied with Unilateral Disuse in the Early Phase

Purpose: It is clinically important to determine the efficacy of estrogen replacement for postmenopausal women combined with mobility difficulties, due to the potential risks of estradiol. The objective of the current study was to investigate the effect of estradiol replacement on osteoporosis induced by the ovariectomy (OVX) combined with unilateral sciatic neurectomy (SN) in a rat model. Method: Female Sprague-Dawley rats were subjected to OVX and unilateral SN on the right hindlimb (OVX+SN) or sham surgery (CTRL). 17β-estradiol (E₂) or vehicle was administrated to the rats immediately, and followed by every other day. Bone mass and trabecular microarchitecture were analyzed using micro-Computed Tomography (micro-CT) and histology at days 3, 7, 14, and 28 post-surgery. The local expressions of sclerostin/SOST, secreted exclusively by osteocytes, and tartrate-resistant acid phosphatase 5b (TRAP 5b), produced mostly by osteoclasts, were examined by immunohistochemistry and TRAP staining, respectively. Serum markers of bone resorption, including C-terminal telopeptides of type I collagen (CTx), receptor activator for nuclear factor κB ligand (RANKL), and TRAP 5b, were quantified by enzyme linked immunosorbent assay (ELISA). Result: Based on micro-CT analysis, E₂ treatment of OVX+SN rats improved the preservation of the bone volume fraction (BV/TV) and trabecular number (Tb.N) in the tibias at day 14 post-surgery, which were 43% and 46% higher in OVX+SN+E₂ rats than those in OVX+SN rats, respectively. However, the impact of E₂ was transient and disappeared at day 28. Expression of sclerostin in the tibias of OVX+SN rats was significantly elevated at day 7 post-surgery compared with the CTRL, but was suppressed until day 14 with E₂ replacement. Conclusion: Our results showed that estrogen replacement could transiently protect against bone loss in OVX rats combined with mechanical unloading. The up-regulation of sclerostin expression appears to be transiently delayed by E₂ treatment in our models.     

Oophorectomy-induced osteopenia in rats in relation to age and time postoophorectomy

Oophorectomized (OVX) rats served for many years as a popular model for 'postmenopausal' osteoporosis in spite of the fact that the rat continues to grow during these experiments. We performed OVX in rats at 1, 3, 6 and 10 months of age and compared the histomorphometric (bone size, bone trabecular and cartilage volume in different areas) and chemical (ash, Ca, P and Mg content) parameters at 2, 6, 8 and 20 weeks post-OVX (2-20 weeks) to those of sham-operated rats. Significant differences were observed only in the animals that were OVX at a young or young-mature age, i.e. mainly at 1 and 3 months and some of the rats at 6 months of age. There were no changes in bone ash and mineral contents in the OVX animals in comparison to sham-operated rats, except 2 weeks post-OVX in the 1-month-old rats where these variables were reduced. The most significant finding was a reduction in the metaphyseal bone volume. This was observed in the rats OVX at 1, 3 and 6 months of age, but not in those OVX at 10 months. In the young OVX rats there was also an increase in epiphyseal cartilage volume. The epiphyseal and diaphyseal bone volumes were not different between the groups at any time postsurgery, explaining the lack of differences in bone ash and mineral contents. There were no significant changes in the results of the histomorphometric studies between OVX and sham-operated rats when surgery was performed at 10 months of age. Since OVX exerts significant changes only in young rapidly growing rats, this approach is an inappropriate model for postmenopausal osteoporosis, which occurs long after bone growth has ended.     

Mast cells accumulate in rat bone marrow after ovariectomy

We have previously shown that mast cells (MC) may act as accessory cells of bone resorption in a synchronized model of resorption. The aim of this study was to verify whether bone marrow MC accumulated during the phase of rapid osteopenia development in estrogen-deprived rats. Sprague-Dawley rats, 90 days old, were subjected to bilateral ovariectomy (OVX) or sham surgery (controls). Bone histomorphometry was performed in the proximal tibia metaphysis. Thirty days after surgery, serum estradiol had dropped (p < 0.002), and osteopenia was well established in the OVX rats (trabecular bone volume decreased by 69%, p < 0.0001). The indices of bone resorption were strongly modified: the number (p < 0.0001) and individual activity (p < 0.001) of the trabecular osteoclasts were prominently increased in the OVX rats. Ovariectomy resulted in a dramatic increase in the number of bone marrow MC (p < 0.0001), 97% of which were of the mucosal subtype. These results show that MC accumulate concomitantly with osteoclast generation in estrogen-deficient rats. Although the biological significance of this MC increase is unknown from these results, we hypothesize that MC might play a role in ovariectomy-induced bone loss similar to that they we previously proposed in a mechanically activated model of resorption.     

Effect of ginseng mixture on osteoporosis in ovariectomized rats

In this study, the authors have characterized the effect of HER-S (red ginseng, Angelicae gigantis Radix, Phyllostachys folium, and soybean extracts) on osteoporosis-associated phenomena in ovariectomized (OVX) rats by measuring body weights and bone histomorphometries in control, sham, OVX, OVX(beta-estradiol-treated), and OVX(HER-S-treated) rats. Light microscopic analyses showed a porous or eroded appearance on the femoral trabecular bone surface in OVX rats, whereas the femoral trabecular bone surfaces of the other groups (control, sham, OVX(17beta-estradiol-treated), and OVX(HER-S-treated) rats) were composed of fine particles. The femoral trabecular bone area and number were decreased in OVX rats, but these reductions were significantly prevented by the administration of HER-S for 7 weeks, similar to estrogen. In the blood biochemistry results, serum phosphorus, calcium, T(3), and T(4) remained unchanged, but blood estrogen levels were significantly increased in HER-S-treated rats, which suggests that estrogen is related to the mechanism of the HER-S-induced antiosteoporosis function in OVX rats.     

卵巢切除及雌二醇对大鼠腰椎松质骨微结构的作用及其生物力学性能的分析

应用micro-CT获得腰椎松质骨微结构的三维参数,分析卵巢切除术与雌二醇干预对大鼠松质骨微结构及整体骨生物力学性能的作用,初步讨论松质骨微结构的改变对生物力学性能的影响.6月龄未交配雌性SD大鼠30只,随机分成3组(每组10只):假手术对照组(Sham)、去卵巢组(OVX)和去卵巢 补充雌二醇组(EBT).术后相同条件饲养5个月,取第3腰椎进行生物力学压缩试验,第4腰椎行micro-CT扫描.结果表明,与相应的Sham组比较,OVX组的BV/TV、Tb.N均明显下降,Tb.Sp和SMI明显增高.EBT组的BV/TV、Tb.N和Tb.Th均大于OVX组,Tb.Th和SMI明显小于OVX组.骨力学性能检测显示OVX组腰椎松质骨E、Fmax和σmax均明显降低,而EBT组上述骨生物力学参数均明显改善.通过micro-CT获得的骨微结构参数并结合骨力学性能检测能为合理评价骨质疏松及抗骨质疏松药物药效研究提供较好的实验依据.     

去卵巢大鼠椎骨组织形态计量学、生物力学特点及相关性研究

采用骨组织形态计量学、骨生物力学和骨矿含量测定等方法研究 10 .5月龄至 16月龄 SD大鼠和双侧卵巢去除术后椎骨的骨质量变化以及它们之间的相关关系。雌性 SD大鼠随机分为 6组 :(1) 10 .5月龄基础组 ;(2 )13月龄假手术组 (sham - 1) ;(3) 13月龄去卵巢组 (OVX- 2 ) ;(4) 16月龄假手术组 (sham - 2 ) ;(5 ) 16月龄去卵巢组(OVX- 2 )和 (6 )雌激素治疗组 :去卵巢 10周后给予雌激素治疗 12周 (OVX- 2 +EE)。所有大鼠在处死前 14、13d和4、3d分别皮下注射四环素和钙黄绿素作体内荧光标记。实验终止时取大鼠第 4腰椎行骨组织形态计量学测量 ,第5腰椎体作试件压缩试验 ,试验后的椎体进行骨矿物元素测定。结果发现 10 .5月龄至 16月龄大鼠椎体骨干重、冠状径、骨小梁面积骨小梁厚度有增加趋势 ,骨的破坏荷载 ,破坏应力和弹性模量也有增加趋势 ,以 13月龄为高峰值。去卵巢 10周后 ,上述指标明显下降 ,2 2周后 ,下降更明显。雌激素治疗后上述指标明显改善。相关研究表明 ,骨小梁面积与骨的破坏应力呈正相关 (P<0 .0 5 ) ,而骨的破坏应力又与骨矿 (主要成分 Ca)含量呈正相关 (P<0 .0 1)。去卵巢后 ,骨小梁面积的下降比骨破坏应力的下降更明显 ,出现的时间更早。本研究表明 ,骨组织形态计量学检测和骨生物力学检