Effect of ovariectomy on BMD, micro-architecture and biomechanics of cortical and cancellous bones in a sheep model

Osteoporotic/osteopenia fractures occur most frequently in trabeculae-rich skeletal sites. The purpose of this study was to use a high-resolution micro-computed tomography (micro-CT) and dual energy X-ray absorptionmeter (DEXA) to investigate the changes in micro-architecture and bone mineral density (BMD) in a sheep model resulted from ovariectomy (OVX). Biomechanical tests were performed to evaluate the strength of the trabecular bone. Twenty adult sheeps were randomly divided into three groups: sham group (n=8), group 1 (n=4) and group 2 (n=8). In groups 1 and 2, all sheep were ovariectomized (OVX); in the sham group, the ovaries were located and the oviducts were ligated. In all animals, BMD for lumbar spine was obtained during the surgical procedure. BMD at the spine, femoral neck and femoral condyle was determined 6 months (group 1) and 12 months (group 2) post-OVX. Lumbar spines and femora were obtained and underwent BMD scan, micro-CT analysis. Compressive mechanical properties were determined from biopsies of vertebral bodies and femoral condyles. BMD, micro-architectural parameters and mechanical properties of cancellous bone did not decrease significantly at 6 months post-OVX. Twelve months after OVX, BMD, micro-architectural parameters and mechanical properties decreased significantly. The results of linear regression analyses showed that trabecular thickness (Tb.Th) (r=0.945, R2=0.886) and bone volume fraction (BV/TV) (r=0.783, R2=0.586) had strong (R2>0.5) correlation to compression stress. In OVX sheep, changes in the structural parameters of trabecular bone are comparable to the human situation during osteoporosis was induced. The sheep model presented seems to meet the criteria for an osteopenia model for fracture treatment with respect to morphometric and mechanical properties. But the duration of OVX must be longer than 12 months to ensure the animal model can be established successfully.     

骨形态计量学观察睾酮对雄性去势大鼠皮质骨的影响

目的通过骨形态计量方法观察雄激素替代疗法对去睾丸大鼠皮质骨代谢的影响。方法30只4月龄SD雄性大鼠,随机分成基础对照组(A组、实验开始时处死),年龄对照组(B组)、去睾丸组(C组)和去睾丸加睾丸酮组(D组),B组和C组生理盐水5ml·kg-1·d-1,D组甲基睾丸酮片1.8mg·kg-1·d-1,灌胃90d。实验结束,处死全部大鼠,取胫骨中段进行不脱钙骨制片,用计算机全自动图象分析系统进行骨组织形态计量学分析。结果去睾丸后皮质骨静态参数如截面总面积、髓腔面积等无明显变化,动态参数骨外膜骨形成降低(P<0.05),内膜骨形成和吸收均有增加趋势。睾酮则使去睾丸大鼠皮质骨的静态参数有增加趋势,促使骨外膜形成增加,减少内膜骨吸收(P<0.05),对内膜骨形成影响不大。结论睾酮补充治疗短期内能对抗去睾丸引起的大鼠皮质骨内外膜的代谢变化,维持正常的皮质骨结构。     

Short-term healing kinetics of cortical and cancellous bone osteopenia induced by unloading during the reloading period in young rats

 We investigated the short-term recuperation of bone mass during skeletal reloading after a period of unloading in young rats. One hind limb of 4-week-old rats was either unloaded irreversibly by sciatic neurectomy, or unloaded reversibly by external fixation. Other animals were sham-operated. After 9 days, the fixation-unloaded limbs were reloaded for 1–3 weeks and were compared with the hind limbs of age-matched unloaded (neurectomized) and sham-operated controls. Cortical and cancellous bone mass was measured using ashing and histomorphometry. Cortical bone mass (expressed as femoral dry and ash weight and tibial cortical bone area) was reduced in both unloaded groups and was accompanied by production of hypomineralized bone, as shown by a reduction in the percent ash of the dry weight. Cancellous bone mass (expressed as bone area and surface at the tibial metaphysis) was also reduced in both unloaded groups. Cortical bone mass deficit was greater in the fixation group than in the neurectomy group. Thereafter it increased in the neurectomy group despite a normal longitudinal growth rate, but returned to age-matched values in the reloaded group by 3 weeks. The changes in tibial cancellous bone mass were more pronounced but followed a similar pattern and normalized by 2 weeks. These data demonstrate that total unloading produced by external fixation causes a greater degree of bone mass deficit than partial unloading (produced by neurectomy); the rate of bone loss during unloading in the rat hind limb is more rapid than its recovery during reloading; and cancellous bone recuperates during the reloading phase faster than does cortical bone. Received: 11 February 1997 / Accepted: 28 May 1997     

Effect of vitamin K deficiency on urinary gamma-carboxyglutamic acid excretion in rats

Since gamma-carboxyglutamic acid (Gla) in Gla-containing proteins is stoichiometrically excreted into urine as free Gla, urinary Gla excretion is believed to reflect the rate of synthesis and degradation of vitamin K-dependent proteins and the utilization of vitamin K in body. We studied the daily changes in urinary Gla excretion and plasma vitamin K-dependent clotting factor levels in rats fed vitamin K-deficient diets followed by subcutaneous injection of vitamin K1 or after the oral administration of Warfarin. Urinary Gla excretion in normal rats fed a standard diet that contained about 500 ng of vitamin K1 per gram of diet was 2.35 +/- 0.25 mumoles/day, but the level in rats fed a markedly vitamin K-deficient diet (less than 5 ng/g) decreased to 1.40 +/- 0.14 mumoles/day. When rats were fed a moderately vitamin K-deficient diet (20-50 ng/g), plasma vitamin K-dependent clotting factor levels decreased significantly, but urinary Gla excretion did not decrease. Warfarin, a vitamin K antagonist, caused a significant decrease in urinary Gla excretion and plasma clotting factor levels. When vitamin K, (200 micrograms/kg) was injected subcutaneously in rats fed a markedly vitamin K-deficient diet, the plasma vitamin K-dependent clotting factor levels recovered quickly to normal, but urinary Gla excretion showed only a partial recovery to 1.74 +/- 0.15 mumoles/day. These results indicate that urinary Gla excretion decreases in vitamin K deficiency, but changes in urinary Gla excretion do not reflect vitamin K deficiency in rats as sensitively as changes in the prothrombin time and plasma K-dependent clotting factor levels.     

Differential effect of vitamin K and vitamin D supplementation on bone mass in young rats fed normal or low calcium diet

The purpose of this study was to clarify the differential effect of vitamin K and vitamin D supplementation on bone mass in young rats fed a normal or low calcium diet. Ninety female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into nine groups with 10 rats in each group: baseline control, and 0.5% (normal) or 0.1% (low) calcium diet, either alone, or with vitamin K (30 mg/100g, food intake), vitamin D (25 micro g/100 g, food intake), or vitamin K + vitamin D. After 10 weeks of feeding, bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. Vitamin K supplementation increased the maturation-related cancellous bone gain and retarded the reduction in the maturation-related cortical bone gain in rats fed a low calcium diet, and increased the maturation-related cortical bone gain in rats fed a normal calcium diet. Vitamin D supplementation reduced the maturation-related cancellous bone gain, prevented the reduction in periosteal bone gain, and enhanced the enlargement of the marrow cavity, with no significant effect on the reduction in the maturation-related cortical bone gain in rats fed a low calcium diet, and increased the maturation- related cancellous and cortical bone gains with increased periosteal bone gain in rats fed a normal calcium diet. An additive effect of vitamin K and vitamin D on the maturation- related cortical bone gain was found in rats fed a normal calcium diet. This study shows the differential effects of vitamin K and vitamin D supplementation on cancellous and cortical bone mass in young rats fed a normal or low calcium diet, as well as the additive effect on cortical bone under calcium sufficient condition.     

Effect of nanostructure on biodegradation behaviors of self-setting apatite/collagen composite cements containing vitamin K2 in rats

Apatite cement and collagen were combined by a mechanochemical method to create a new self-setting apatite/collagen composite cement, and menatetrenone (VK2) was loaded into a drug-delivery system to test biocompatibility in rats. Powder X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), and electron probe microanalyzer (EPMA) were performed to characterize the physicochemical properties of apatite/collagen composite cements. The XRD results suggested that ground apatite/collagen cement was completely transformed into bone-like hydroxyapatite, but that without grinding was incomplete. The SEM and EPMA results suggested that ground apatite/collagen cement was homogeneously dispersed of nanoapatite crystals in collagen matrices, similar to that in natural bone. In contrast, the cement without grinding was heterogeneously distributed. To evaluate in-vivo cement density (CMM), microradiograms were measured for 72 days after implanting apatite/collagen composite cements in intramuscular tissue on the backs of rats, and cross sections of the cements and surrounding soft tissues were observed by microscope. The CMM results of the apatite/collagen composite cements suggested that the biodegradation rate was dependent on the cement quality and nanogeometrical structure. The CMM result of VK2-loaded apatite/collagen cements suggested that the biodegradation rates of the cements were significantly dependent on their formulation. The CMM of ground apatite/collagen cement increased until 7 days and then decreased, and bone-like cells penetrated deeply in the center. The microphotograph and CMM results of apatite/collagen without grinding indicated that a lot of bone-like cells penetrated into the cement and the cement shape was totally deformed.     

Effect of chronic vitamin E deficiency on the synapses of cerebellar glomeruli in young rats

A morphometric investigation has been carried out on the synaptic junctions in the cerebellar glomeruli of young-adult rats chronically deprived of vitamin E for 10 months and control animals of the same age. The following parameters were evaluated: the average length of the synapses (L), the numerical (NV) as well as the surface (SV) density of the synaptic contact zones. The results from these experimental groups were compared with data from young, adult and old rats. The results obtained show a significant decrease of the surface density of the synaptic contact zones in old and alpha-tocopherol deprived young-adult (11-month-old) rats as compared to younger and normally fed animals. This reduction of the synaptic contact area seems to be due to the marked decline in the number of synapses found in both cases. The average size (L) of the synaptic junctions, on the other hand, was increased in alpha-tocopherol deficient rats as compared to normally fed littermates. The significant reduction of the synaptic contact area in old and vitamin E deprived young rats supports the hypothesis that a common denominator may be responsible to explain this alteration. Because of the recognized protective role of alpha-tocopherol against free radical attacks on plasma membranes, the present findings support an involvement of membrane structural alterations in aging as well as in vitamin E deficiency.     

Effect of irradiation on vitamin status and spermatogenesis in rats

It is shown that in rats exposed to single external irradiation in a dose of 0.5 Gy the concentration of spermatozoa surpasses that of nonirradiated controls. The level of vitamin B₂ increases in the liver and testes of irradiated animals. For the given dose of ionizing radiation, the participation of angiotensin-converting enzyme in cell maturation during spermatogenesis is not observed. Various changes in the content of antioxidant vitamins at different stages of the experiment are described. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 524–527, May, 1997     

Effects of vitamin K2 on the development of osteopenia in rats as the models of osteoporosis

Vitamin K2 is widely used for the treatment of osteoporosis in Japan. To understand the effects of vitamin K2 on bone mass and bone metabolism, we reviewed its effects on the development of osteopenia in rats, which characterizes models of osteoporosis. Vitamin K2 was found to attenuate the increase in bone resorption and/or maintain bone formation, reduce bone loss, protect against the loss of trabecular bone mass and its connectivity, and prevent the decrease in strength of the long bone in ovariectomized rats. However, combined treatment of bisphosphonates and vitamin K2 had an additive effect in preventing the deterioration of the trabecular bone architecture in ovariectomized rats, while the combined treatment of raloxifene and vitamin K2 improved the bone strength of the femoral neck. The use of vitamin K2 alone suppressed the increase in trabecular bone turnover and endocortical bone resorption, which attenuated the development of cancellous and cortical osteopenia in orchidectomized rats. In addition, vitamin K2 inhibited the decrease in bone formation in prednisolone-treated rats, thereby preventing cancellous and cortical osteopenia. In sciatic neurectomized rats, vitamin K2 suppressed endocortical bone resorption and stimulated bone formation, delaying the reduction of the trabecular thickness and retarding the development of cortical osteopenia. Vitamin K2 also prevented the acceleration of bone resorption and the reduction in bone formation in tail-suspended rats, which counteracted cancellous bone loss. Concomitant use of vitamin K2 with a bisphosphonate ameliorated the suppression of bone formation and more effectively prevented cancellous bone loss in tail-suspended rats. Vitamin K2 stimulated renal calcium reabsorption, retarded the increase in serum parathyroid hormone levels, and attenuated cortical bone loss primarily by suppressing bone resorption in calcium-deficient rats while maintaining the strength of the long bone in rats with magnesium deficiency. These findings suggest that vitamin K2 may not only stimulate bone formation, but may also suppress bone resorption. Thus, vitamin K2 could regulate bone metabolism in rats, which represented the various models of osteoporosis. However, the effects of vitamin K2 on bone mass and bone metabolism seem to be modest.     

Structure and mineral content in weight-bearing bones following hindlimb suspension in young rats.

Responses of structure and mineral content in weight-bearing bones to 1-week hindlimb suspension were studied in growing rats. The weight, volume, and length of femur, tibia, and fibula were less in suspended rats than cage controls, although there was no significant difference in the weight/volume ratio. The diameter of mid-shaft, the thickness of cortical bone, and the ratio of cortical bone thickness to femur diameter were significantly less in the suspended rats. The width of marrow space showed no differences, suggesting that the cortical bone was mainly affected. The sum of mineral content and the mineral content per diameter in the cross section of femurs were decreased following suspension, suggesting a change in the internal structure of cortical bone. Data also indicated that the reduction of femur diameter was caused by decreased thickness which is accompanied by the decreased mineral content of the cortical bone. It was suggested that there is a close relationship between the responses of bones and muscles to hindlimb suspension of young rats, although the degree of weight loss was greater in muscles than in bones.     

Effect of bradykinin and morphine on sensomotor cortical neurons in rats

Acute experiments on rats showed that bradykinin, injected by microiontophoresis, activates sensomotor cortical neurons in rats. Morphine, administered in the same way, prevents the development of the bradykinin effect. Bradykinin, it is suggested, acts on opiate receptors in cerebral cortical neurons.Laboratory of Pharmacology of the Nervous System, Institute of Pharmacology, Academy of Medical Sciences of the USSR. Laboratory of Emotions and Emotional Stresses, P. K. Anokhin Institute of Normal Physiology, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 10, pp. 450–452, October, 1978.     

维生素K对糖代谢的影响

近期研究显示维生素K在控制血糖方面能起到积极的作用.摄入较低剂量维生素K1的小鼠比高维生素K1饮食的小鼠血糖高,且其胰岛素的释放高峰较后者延迟.临床试验也证实健康年轻男性短时间补充维生素K能改善胰岛素的作用及促进外周组织对血糖的代谢.对于维生素K影响血糖代谢的潜在机制尚未完全明了,但近期研究显示可能与骨钙素活性有关.     

Experimental study of the antiamnesic activity of piracetam in orchidectomized rats

All-Union Research Center for Safety of Biologically Active Substances, Staraya Kupavna, Moscow Region. (Presented by Academician of the Academy of Medical Sciences of the USSR P. V. Sergeev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 3, pp. 290–291, March, 1992.     

Effect of gossypol on the thyroid in young rats

The effect of gossypol on thyroid structure and function was examined. Groups of young male rats were subcutaneously injected daily with 0.2 ml gossypol acetic acid in doses of 1, 5, or 10 mg per kg body weight. Control rats were injected with vehicle only. At the end of the 15-day treatment, there was a dose-dependent decrease in the serum concentrations of thyroid hormones, i.e., free thyroxine, triiodothyronine, and reverse triiodothyronine. Histopathological examination revealed focal degeneration of thyroid follicles after a dose of 1 mg gossypol per kg body weight; widespread follicular atrophy occurred with 5 mg and 10 mg gossypol per kg body weight. The degenerating follicles were lined with discontinuous cuboidal or squamous follicular epithelium, some of which exfoliated into colloid.     

泼尼松对大鼠松质骨结构的影响

目的：探讨泼尼松对松质骨结构的影响。方法：用泼尼松４．５ｍｇ／ｋｇ,给３月龄ＳＤ雄性大鼠灌胃,每周２次,持续９０ｄ。扫描电镜观察大鼠腰椎松质骨结构的改变。结果：与正常组比较,泼尼松组的大鼠骨小梁变少、变细、断裂、连接不紧密,表现常见骨吸收形成的陷窝。结论：糖皮质激素可造成松质骨三维结构的损害,使力学强度降低,增加骨折的危险性。     

Effect of body vitamin K level on collagen metabolism in the skin

In rats with secondary avitaminosis-K induced by Pelentan the collagen content in the skin is reduced and the content of free hydroxyproline increased. The rate of acid hydrolysis of collagen is increased. Vitamin K (Vikasol^*) prevents the development of the changes in collagen metabolism.Department of Biochemistry, Izhevsk Medical Institute.(Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 6, pp. 665–666, June, 1976.