Increased3H-uridine levels in osteocytes following a single short period of dynamic bone loadingin vivo

Summary Both ulnas of skeletally mature roosters (Gallus domesticus) were deprived of functional load bearing by proximal and distal submetaphyseal osteotomies. Twenty-four hours later the animals were injected with 1.5 mCi of³H-uridine and the ulna on one side was subjected to a single period of a cyclical load engendering physiological strain levels at 1 Hz for 6 min. Twenty-fours after loading the animals were killed. Autoradiographic examination of comparable regions of cortex in sections from the bones' midshafts showed that in the loaded bones, 72±2.7% of osteocytes were labeled compared with 12±3.5% in the corresponding areas of their contralateral nonloaded pair (P<0.001). The number of grains per labeled osteocyte was also higher in the loaded side (6±0.5 compared with 4±0.5,P<0.01). There was no obvious correlation between the longitudinal strain distribution during artificial loading and the distribution of labeled osteocytes throughout the bone cross-section. However, previous long-term experiments using a similar loading preparation had consistently shown the site of most periosteal new bone formation to also not be directly related to the local strain magnitude. Perhaps it is significant that the greatest percentage of labeled cells were found in the cortex where the long-term experiments had shown most new bone formation to subsequently occur.     

可塑形组织工程骨修复兔颅骨缺损的组织学及力学研究

目的探讨用藻酸钙凝胶、成骨细胞和骨粉复合构建可塑形组织工程骨修复兔颅骨缺损后,体内成骨的组织学及生物力学特征。方法28只日本大耳白兔,随机分为A组(16只)、B组(8只)和C组(4只)。制备兔颅骨左右两侧直径1cm的骨膜-颅骨全层缺损,左侧用藻酸钙凝胶-成骨细胞-骨粉填补修复为A1组(n=16);右侧用藻酸钙凝胶-骨粉填补修复为A2组(n=16);B组骨缺损不作处理,为空白对照组(n=16);C组为正常组。术后6周和12周时,行大体观察及组织学观察;12周时行生物力学测试。结果术后6、12周时,A1组:颅骨缺损基本被硬组织所修复,镜下见材料已大部分被骨组织替代,成骨面积为40.92%±19.36%;A2组:材料部分被骨组织替代,成骨面积为18.51%±6.01%;B组:颅骨缺损边缘可见硬组织形成,镜下见修复组织以致密纤维组织为主,成骨面积为12.72%±9.46%。术后12周,生物力学测试修复组织能耐受的最大压力载荷,A1组37.33±2.95N;A2组30.59±4.65N;B组29.5±2.05N;C组41.55±2.52N;A1组明显大于A2组和B组(P<0.05)。最大载荷时应变位移,A1组1.05±0.20mm;A2组1.35±0.44mm;B组1.57±0.31mm;C组0.95±0.17mm;A1组小于B组(P<0.05)。载荷/应变比值,A1组35.82±6.48N/mm;A2组24.95±12.40N/mm;B组19.90±5.47N/mm;C组47.57±11.22N/mm;A1组大于B组(P<     

Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts.

Mechanical loading presents a potent osteogenic stimulus to bone cells, but bone cells desensitize rapidly to mechanical stimulation. Resensitization must occur before the cells can transduce future mechanical signals effectively. Previous experiments show that mechanical loading protocols are more osteogenic if the load cycles are divided into several discrete bouts, separated by several hours, than if the cycles are applied in a single uninterrupted bout. We investigated the effect of discrete mechanical loading bouts on structure and biomechanical properties of the rat ulna after 16 weeks of loading. The right ulnas of 26 adult female rats were subjected to 360 load cycles/day, delivered in a haversine waveform at 17 N peak force, 3 days/week for 16 weeks. One-half of the animals (n = 13) were administered all 360 daily cycles in a single uninterrupted bout (360 x 1); the other half were administered 90 cycles four times per day (90 x 4), with 3 h between bouts. A nonloaded baseline control (BLC) group and an age-matched control (AMC) group (n = 9/group) were included in the experiment. The following measurements were collected after death: in situ mechanical strain at the ulna midshaft; ulnar length; maximum and minimum second moments of area (I(MAx) and I(MIN)) along the entire length of the ulnas (1-mm increments); and ultimate force, energy to failure, and stiffness of whole ulnas. Qualitative observations of bone morphology were made from whole bone images reconstructed from microcomputed tomography (microCT) slices. Loading according to the 360 x 1 and 90 x 4 schedules improved ultimate force by 64% and 87%, energy to failure by 94% and 165%, I(MAX) by 13% and 26% (in the middistal diaphysis), I(MIN) by 69% and 96% (in the middistal diaphysis), and reduced peak mechanical strain by 40% and 36%, respectively. The large increases in biomechanical properties occurred despite very low 5-12% gains in areal bone mineral density (aBMD) and bone mineral content (BMC). Mechanical loading is more effective in enhancing bone biomechanical and structural properties if the loads are applied in discrete bouts, separated by recovery periods (90 x 4 schedule), than if the loads are applied in a single session (360 x 1). Modest increases in aBMD and BMC can improve biomechanical properties substantially if the new bone formation is localized to the most biomechanically relevant sites, as occurs during load-induced bone formation.     

Deletion of a Single β‐Catenin Allele in Osteocytes Abolishes the Bone Anabolic Response to Loading

The Wnt/β‐catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β‐catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18‐ to 24‐week‐old osteocyte β‐catenin haploinsufficient mice (Dmp1‐Cre × β‐catenin fl/ + ; HET cKO) were compared with their β‐catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µ?, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro‐computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone‐formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β‐catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β‐catenin is required for bone formation in response to mechanical loading. © 2014 American Society for Bone and Mineral Research     

Validation of a technique for studying functional adaptation of the mouse ulna in response to mechanical loading

Functional adaptation of the mouse ulna in response to artificial loading in vivo was assessed using a technique previously developed in the rat. Strain gauge recordings from the mouse ulnar midshaft during locomotion showed peak strains of 1680 muepsilon and maximum strain rates of 0.03 sec(-1). During falls from 20 cm these reached 2620 muepsilon and 0.10 sec(-1). Axial loads of 3.0 N and 4.3 N, applied through the olecranon and flexed carpus, engendered peak strains at the lateral ulnar midshaft of 2000 muepsilon and 3000 muepsilon, respectively. The left ulnae of 17, 17-week-old female CD1 mice were loaded for 10 min with a 4 Hz trapezoidal wave engendering a strain rate of 0.1 sec(-1) for 5 days/week for 2 weeks. The mice were killed 3 days later. The response of the cortical bone of the diaphysis was assessed histomorphometrically using double calcein labels administered on days 3 and 12 of the loading period. Loading to peak strains of 2000 muepsilon stimulated lamellar periosteal bone formation, but no response endosteally. The greatest increase in cortical bone area was 4 mm distal to the midshaft (5 +/- 0.4% compared with 0.1 +/- 0.1% in controls [p < 0.01]). Periosteal bone formation rate (BFR) at this site was 0.73 +/- 0.06 microm(2)/microm per day, compared with 0.03 +/- 0.02 microm(2)/microm per day in controls (p < 0.01). Loading to peak strains of 3000 muepsilon induced a mixed woven/lamellar periosteal response and lamellar endosteal bone formation. Both of these were greatest 3-4 mm distal to the ulnar midshaft. At this level, the loading-induced periosteal response increased cortical bone area by 21 +/- 4% compared with 0.03 +/- 0.02% in controls, and resulted in a BFR of 2.84 +/- 0.42 microm(2)/microm per day, compared with 0.01 +/- 0.01 microm(2)/microm per day in controls (p < 0.05). Endosteal new bone formation resulted in a 2 +/- 0.4% increase in cortical bone area, compared with 0.4 +/- 0.3% in controls, and a BFR of 1.05 +/- 0.23 microm(2)/microm per day, compared with 0.22 +/- 0.15 microm(2)/microm per day in controls (p < 0.05). These data show that the axial ulna loading technique developed in the rat can be used successfully in the mouse. As in the rat, a short daily period of loading results in an osteogenic response related to peak strain magnitude. One important advantage in using mice over rats involves the potential for assessing the effects of loading in transgenics.     

Functional Adaptation to Loading of a Single Bone Is Neuronally Regulated and Involves Multiple Bones

Regulation of load‐induced bone formation is considered a local phenomenon controlled by osteocytes, although it has also been hypothesized that functional adaptation may be neuronally regulated. The aim of this study was to examine bone formation in multiple bones, in response to loading of a single bone, and to determine whether adaptation may be neuronally regulated. Load‐induced responses in the left and right ulnas and humeri were determined after loading of the right ulna in male Sprague‐Dawley rats (69 ± 16 days of age). After a single period of loading at ?760‐, ?2000‐, or ?3750‐μ? initial peak strain, rats were given calcein to label new bone formation. Bone formation and bone neuropeptide concentrations were determined at 10 days. In one group, temporary neuronal blocking was achieved by perineural anesthesia of the brachial plexus with bupivicaine during loading. We found right ulna loading induces adaptive responses in other bones in both thoracic limbs compared with Sham controls and that neuronal blocking during loading abrogated bone formation in the loaded ulna and other thoracic limb bones. Skeletal adaptation was more evident in distal long bones compared with proximal long bones. We also found that the single period of loading modulated bone neuropeptide concentrations persistently for 10 days. We conclude that functional adaptation to loading of a single bone in young rapidly growing rats is neuronally regulated and involves multiple bones. Persistent changes in bone neuropeptide concentrations after a single loading period suggest that plasticity exists in the innervation of bone.     

Low-magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle.

Strategies to counteract bone loss with exercise have had fairly limited success, particularly those regimens subjecting the skeleton to mild activity such as walking. In contrast, here we show that it is possible to induce substantial bone formation with low-magnitude loading. In two distinct in vivo models of bone adaptation, we found that insertion of a 10-s rest interval between each load cycle transformed a locomotion-like loading regime that minimally influenced osteoblast activity into a potent anabolic stimulus. In the avian ulna model, the minimal mean (+SE) periosteal labeled surface (Ps.LS) observed in the intact contralateral bones (1.6 +/- 1.5%) was doubled after 3 consecutive days of low-magnitude loading (3.8 +/- 1.5%; p = 0.03). However, modifying the regimen by inserting 10 s of rest between each load cycle significantly enhanced the periosteal response (21.9 +/- 4.5%; p = 0.03). In the murine tibia model, 5 consecutive days of 100 low-magnitude loading cycles did not significantly alter mean periosteal bone formation rate (BFR) compared with contralateral bones (0.011 +/- 0.005 microm3/microm2 per day vs. 0.021 +/- 0.013 microm3/microm2 per day). In contrast, separating each of 10 of the same loading cycles with 10 s of rest significantly elevated periosteal BFR (0.167 +/- 0.049 microm3/microm2 per day; p = 0.01). Endocortical bone formation parameters were not altered by any loading regimen in either model. We conclude that 10 s of rest between each load cycle of a low-magnitude loading protocol greatly enhances the osteogenic potential of the regimen.     

Dichloroacetate increases skeletal muscle pyruvate dehydrogenase activity during acute limb ischemia

The purpose of this study was to evaluate the effects of dichloroacetate sodium (DCA), a drug that inactivates pyruvate dehydrogenase kinase (PDH-K), on pyruvate dehydrogenase (PDH) activity, lactate level, and function of skeletal muscle in an experimental model of acute limb ischemia. Thirty-two male Sprague-Dawley rats underwent right iliac artery ligation to produce hindlimb ischemia. After 2 hours of ischemia, 16 animals received intravenous DCA (15 mg/100 g body weight) and 16 control animals received an equivalent volume of normal saline. After an additional 1 hour of ischemia (total 3 hours) tibialis anterior muscle from the ischemic limb and contralateral nonischemic limb was excised, rapidly freeze-clamped with Wallenberg tongs cooled in liquid nitrogen, and stored at -70 degrees C. Muscles specimens were subsequently assayed for PDH activity and lactate level by use of spectrophotometric techniques. An additional 16 animals (DCA-treated, n = 8; control, n = 8) underwent ex-vivo gastrocnemius muscle fatigue testing with a 10 g tension preload after 3 hours of limb ischemia. In ischemic hind limbs, DCA treatment significantly (p = 0.025) increased PDH activity (19.6 +/-1.6 micromol/min/g dry weight) compared to controls (13.1 +/-1.3 micromol/min/g dry weight). DCA treatment did not increase (p = 0.13) skeletal muscle PDH activity in the nonischemic limbs (9.6 +/-1.1 micromol/min/g dry weight, controls; 13.2 +/-1.3 micromol/min/g dry weight, DCA group). In DCA-treated animals, hind limb ischemia resulted in no significant increase in muscle lactate levels compared to the nonischemic limb, while control animals demonstrated a significant (p = 0.005) elevation in lactate level in ischemic limbs compared to contralateral nonischemic limb. Ischemia induced a significant decrease in time to muscle fatigue in both DCA-treated and control animals (p = 0.002 and 0.001, respectively). Time to muscle fatigue in DCA-treated animals was increased compared to controls (2.6 +/-0.3 versus 2 +/-0.6 minutes; p < 0.05)in ischemic limbs but was not significantly different in nonischemic limbs (DCA = 3.3 +/-0.5 minutes; control = 3.1 +/-0.6 minutes). Treatment with DCA during acute limb ischemia reduced the depression of PDH activity and lactate level of skeletal muscle. Ischemic muscle function was also improved by DCA treatment. Further investigation of the potential beneficial effects of DCA treatment on muscle injury during ischemia and reperfusion is warranted.     

Osteocytic expression of constitutive NO synthase isoforms in the femoral neck cortex: a case-control study of intracapsular hip fracture.

NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25-35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack. INTRODUCTION: Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone. MATERIALS AND METHODS: Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68-91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured. RESULTS: The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 +/- 1.49, controls: 56.68 +/- 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 +/- 1.53, controls: 56.47 +/- 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 +/- 2.2 microm, cases: 61.7 +/- 2.0 microm; p < 0.0001; nNOS: controls: 52.4 +/- 1.7 microm, cases: 60.2 +/- 2.1 microm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 +/- 4.0 microm (p = 0.0012). CONCLUSION: The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.     

固定一侧前肢后对犬后肢负重的影响

目的 建立三足负重犬模型,模仿人的髋关节应力,观察其双后肢的动力学参数变化. 方法 选用10 只健康成年 Beagle 犬,雌雄不限,体重20～25 kg,一侧腕关节屈曲90°位固定,建立三足负重犬模型.于固定前后(分别为对照组和实验组)行步态分析,比较双后肢的触地时间和地面反力变化. 结果 犬固定一侧前肢后,可三足行走,步态发生明显变化,呈前半身抬起的跳跃状行走,双后肢交替负重前行.实验组双后肢的站立相时间均有不同程度增加,同侧后肢为 (0.48±0.04)s,对侧后肢为(0.46±0.06)s,但与对照组犬的(0.43±0.05) s 比较无统计学意义(P>0.05).实验组双后肢峰值垂直地面反力明显增加,与对照组(3.26±0.48)倍体重比较差异有统计学意义(P<0.05).实验组同侧后肢的峰值加速力为 (0.80±0.30)倍体重,与对照组犬(0.72±0.13)倍体重比较差异有统计学意义(P<0.05);对侧后肢为(0.68±0.22)倍体重,与对照组比较无统计学意义(P>0.05);实验组双后肢的峰值减速力明显减小,同侧后肢为- (0.26±0.14)倍体重,对侧后肢为- (0.13±0.05) 倍体重,与对照组- (0.43±0.13) 倍体重比较差异有统计学意义(P<0.05).对照组犬的主要负重肢体为前肢,前肢承担体重的 62.8%±2.4%,后肢承担 37.2%±1.8%,实验组犬的主要负重肢体为后肢,承担体重的59.1±6.7%. 结论 三足负重犬可作为一个负重动物模型用来研究生物力学因素在髋关节相关疾病中的作用.     

Determinants of peak trabecular bone density in women: the role of androgens, estrogen, and exercise

To elucidate determinants of peak trabecular bone density, we studied the role of androgens, estrogen, and aerobic exercise in 30 women from 18 to 22 years old. The women were divided into three groups: Sedentary, 11 normal women who did not exercise regularly; eumenorrheic, 10 athletes with normal menstrual function; and oligomenorrheic, 9 athletes with exercise-induced oligomenorrhea. All athletes participated in aerobic sports that did not involve selective resistance loading of the back. Serum free and albumin-bound testosterone (fab T), androstenedione (A), and estradiol (E2) were measured on four separate occasions at consecutive 7 day intervals and averaged. Trabecular density was measured by quantitative computed tomography of the lumbar spine. Peak trabecular bone density was related to fab T (r = 0.48, p = 0.007), A (r = 0.40, p = 0.03), and E2 (r = 0.40, p = 0.04). When taken in combination, androgens and estrogen each accounted independently for significant portions of the variance in bone density [fab T and E2 (R2 = 0.38, p = 0.002) and A and E2 (R2 = 0.27, p = 0.01)]. Bone density (mg/ml, mean +/- standard error of the mean, SEM) in the sedentary group (174 +/- 6) was not significantly different from that in the eumenorrheic (183 +/- 12, p = 0.47) or oligomenorrheic (161 +/- 11, p = 0.32) subjects. We conclude that androgens and estrogen function as independent and additive determinants of peak trabecular bone density in young women. The quantitative impact of aerobic exercise (without resistance loading) and exercise-induced menstrual dysfunction appears to be less important than that of the hormones.     

Risedronate and alendronate suppress osteocyte apoptosis following cyclic fatigue loading

PURPOSE: The purpose of this study was to determine whether bisphosphonate treatment can prevent or delay osteocyte apoptosis in a cyclic fatigue animal model and if there are differences between two different bisphosphonates in their effects on osteocyte apoptosis. INTRODUCTION: Fatigue loading induces microdamage in long bones in rats and causes osteocyte apoptosis. In vitro data suggest that the bisphosphonates can prevent osteocyte apoptosis. MATERIALS AND METHODS: Six month old female Sprague-Dawley rats (n=72) were given a daily subcutaneous (sc) injection of saline vehicle, risedronate (RIS: 0.05 mug/kg per day) or alendronate (ALN: 0.1 mug/kg per day). On the 8th day of drug treatment, an axial compressive load was applied to the right ulna using a load-controlled electromagnetic device (17N, 6000 cycles, 2 Hz, 10% loss of stiffness approximately 1 h). Three, seven or ten days after loading, the animals were sacrificed. Immunohistochemistry for caspase-3 was performed to assess the extent of osteocyte apoptosis in loaded and non-loaded ulnas. RESULTS: Microdamage (Mdx) created by cyclic loading of the ulna induced a significant increase (p=0.03) in the number of apoptotic osteocytes compared to non-damaged regions of the same ulna, and compared to the contralateral non-loaded ulna. Risedronate and alendronate had an early effect (3 days after loading) on reducing load-induced osteocyte apoptosis. Risedronate significantly reduced the density of apoptotic osteocytes compared to vehicle-treated controls by approximately 50% in the Mdx area, whereas alendronate reduced it by approximately 40%. There were no differences among groups by seven days following loading. CONCLUSIONS: (1) Low doses of risedronate or alendronate suppressed osteocyte apoptosis induced by fatigue loading of the ulna in rats. (2) There was no difference between the effects of risedronate or alendronate on osteocyte apoptosis at these doses.     

Effects of Deletion of ERα in Osteoblast‐Lineage Cells on Bone Mass and Adaptation to Mechanical Loading Differ in Female and Male Mice

Estrogen receptor alpha (ERα) has been implicated in bone's response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed‐background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell–specific ERαKO mice. Therefore, we crossed ERα floxed (ERα^fl/fl) and osteocalcin‐Cre (OC‐Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC‐ERαKO) and littermate controls (LC). At 10 weeks of age, the left tibia was loaded in vivo for 2 weeks. We analyzed bone mass through micro‐CT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared with LC, female pOC‐ERαKO mice had decreased cortical and cancellous bone mass, whereas male pOC‐ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC‐ERαKO female L₅ vertebrae and with increased maximum moment in pOC‐ERαKO male femora. Female pOC‐ERαKO mice responded more to mechanical loading, whereas the response of pOC‐ERαKO male animals was similar to their littermate controls. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.     

Estrogen receptor beta gene polymorphisms are associated with higher bone mineral density in premenopausal,but not postmenopausal southern Chinese women

Bone mineral density (BMD), the main determining risk factor for osteoporotic fractures, has a strong genetic component. Estrogen and its receptors play a critical role in both skeletal maturity and bone loss. We investigated the association between dinucleotide (cytosine-adenine; CA) repeat polymorphisms located in the flanking region of the estrogen receptor beta gene and bone mineral density (BMD) in 325 healthy southern Chinese women. BMD at the lumbar spine and hip region were measured using dual-energy X-ray absorptiometry (DEXA). The number of the repeats observed in our population ranged from 16 to 28. After adjusting for age, height, weight, and years of estrogen exposure, we observed that premenopausal subjects (n = 120) bearing at least one allele of 20 CA repeats had significantly higher BMD at the L2-4 lumbar spine (1.049 +/- 0.016 vs. 0.984 +/- 0.015; p = 0.01), total hip (0.836 +/- 0.014 vs. 0.813 +/- 0.013; p < 0.02), femoral neck (0.773 +/- 0.014 vs. 0.728 +/- 0.013; p = 0.02), trochanter (0.665 +/- 0.013 vs. 0.614 +/- 0.012; p = 0.01), and Ward's triangle (0.715 +/- 0.017 vs. 0.651 +/- 0.016; p = 0.02). There was no difference in the vertebral area of L-3 and femoral neck width in these premenopausal women with or without 20 CA repeats. However, in postmenopausal women (n = 205), Estrogen receptor beta (ER beta) gene polymorphisms were not related to BMD at any skeletal site. We conclude that ER beta gene polymorphisms are associated with higher BMD in premenopausal women, suggesting that the ER beta gene may have a modulatory role in bone metabolism in young adulthood.     

Femoral vein ligation increases bone mass in the hindlimb suspended rat

Bone remodeling in response to changing mechanical demands is well recognized. It has been hypothesized that alterations in interstitial fluid flow (IFF), due to intraosseous pressure changes, influence bone remodeling. The goal of this study was to investigate the role of IFF in bone in the absence of mechanical strain using an in vivo model, the hindlimb suspended rat. Bone remodeling was assessed by direct measurements of weight, dimensions, bone mineral content (BMC) and bone mineral density (BMD) by dual-energy X-ray absorptiometry (DEXA), and trabecular density using peripheral computed tomography (pQCT). Ligation of one femoral vein was performed as a means to alter the IFF within the ipsilateral femur; the contralateral limb was sham-operated as control. Animals were suspended for a period of 19 days. Intramedullary pressure in the venous-ligated femurs increased relative to the sham-operated control femurs (27.8 mmHg vs. 16.4 mmHg, p < 0.05), suggesting venous ligation increased IFF proportional to the pressure drop across the bone. Bone mineral content (BMC), when normalized to body weight, increased significantly in the venous-ligated femurs relative to control limbs (115.9 +/- 15.6% vs. 103.8 +/- 13.2%, p < 0.001); similarly, gains in length (106.2 +/- 2.4% vs. 104.5 +/- 2.1%, p < 0.05) and distal width (110.8 +/- 10.3% vs. 106.2 +/- 8.2%, p < 0.05) for the femurs with venous ligation were significantly greater relative to sham control. Furthermore, trabecular density was significantly higher in the femurs with venous ligation (351 +/- 12 g/cm3 vs. 329 +/- 11 g/cm3, p < 0.05). Daily administration of the cyclooxygenase inhibitor, indomethacin, via drinking water, suppressed the length increases observed for the venous ligated femur, suggesting a role for prostaglandins in IFF-mediated remodeling. These results suggest that IFF can directly influence bone adaptation independent of mechanical loading, and supports the hypothesis that fluid flow modulates bone remodeling.     

Uniaxial yield strains for bovine trabecular bone are isotropic and asymmetric.

Although evidence suggests that yield strains for trabecular bone are isotropic, i.e., independent of loading direction, decisive support for this hypothesis has remained elusive. To explicitly test whether yield strains for trabecular bone are isotropic, compressive and tensile yield strains of 51 specimens of bovine tibial trabecular bone (0.41 +/- 0.08 g/cm3 [mean apparent density +/- SD]) were measured without end artifacts in on-axis (along the principal trabecular orientation) and off-axis (30-40 degrees oblique to on-axis) orientations. Yield strains for the on-axis and off-axis orientations were similar in tension (0.80 +/- 0.03% compared with 0.85 +/- 0.04%, p = 0.21) and compression (0.97 +/- 0.05% compared with 0.96 +/- 0.07%, p > 0.99); as expected, modulus and strength depended on loading direction. When considered with an ancillary experiment on bovine tibial trabecular bone that showed yield strains to be similar between on-axis and 90 degrees off-axis bone, these results firmly establish the isotropy of uniaxial yield strains for bovine tibial trabecular bone. This bone is of high density and has a strong, plate-type, anisotropic architecture. Therefore, yield strains for uniaxial loading are expected to be isotropic, or nearly so, for other types of dense trabecular bone, although further work is required to confirm this and to establish this behavior for bone of lower density.     

Elastic anisotropy and collagen orientation of osteonal bone are dependent on the mechanical strain distribution.

There is evidence that the collagen microarchitecture of bone is influenced by mechanical stresses or strains. We hypothesized that peak functional strains correlate with the elastic anisotropy and collagen orientation of bone tissue and that the bone anisotropy might be changed by altering the strain patterns in canine radii for 12 months. We tested these hypotheses in studies using nine adult foxhounds. The baseline group (n = 3) had three rosette strain gauges placed around the midshaft of the radius, and strain distributions were measured during walking. The osteotomy group (n = 3) had 2 cm of the ulna surgically removed, and the sham group (n = 3) received a sham osteotomy. The osteotomy and sham groups were allowed free movement in cages with runs for 12 months, after which strain distributions were measured on the radii during walking. Bone-tissue anisotropy and collagen architecture were measured in radii from which the in vivo longitudinal strain patterns had been measured. The collagen birefringence patterns were measured with use of a circularly polarized light technique, and the elastic anisotropy of the bone, mineral, and collagen matrix was evaluated with a novel acoustic microscopy technique. Peak longitudinal strains in the radius correlated with the normalized longitudinal structure index (a polarized light measure of collagen birefringence) and the tissue anisotropy ratio. The average anisotropy ratio was 1.28+/-0.01 in the posterior (compressive) cortex and 1.43+/-0.01 in the anterior (tensile) cortex (these values are significantly different at p < 0.0001). The ulnar osteotomy changed the strain pattern on the radius, causing increased tensile strains in the medial cortex by more than 5-fold that were associated with a significant increase in the anisotropy ratio in the bone tissue. The longitudinal structure index was strongly correlated (r = 0.62, p < 0.005) with the anisotropy ratio of demineralized bone but was not correlated with that of deproteinized bone; this indicates that it reflects collagen fibril orientation in the bone matrix. These results indicate that mechanical strains affect both collagen and mineral microarchitecture in bone tissue, i.e., tensile strains are associated with increased tissue anisotropy and compressive strains, with decreased anisotropy.     

Loading-related increases in prostaglandin production in cores of adult canine cancellous bone in vitro: a role for prostacyclin in adaptive bone remodeling?

Cyclic mechanical loading sufficient to engender strains of physiologic magnitude applied to recently excised canine cancellous bone cores in vitro increased the release of prostaglandin E (PGE) and prostacyclin (PGI2, measured as its breakdown product 6-keto-PGF1 alpha), during a 15 minute loading period in which PG levels were measured in perfusing medium at 5 minute intervals. Peak production occurred in the 0-5 minute sample. Mean levels preload compared to during load were PGE, 2.66 and 3.67 ng/ml (p less than 0.002); and 6-keto-PGF1 alpha, 543 and 868 pg/ml (p less than 0.007). The elevated levels then declined to preload levels during the loading period. However, the 5-10 minute but not the 10-15 minute samples still contained levels greater than preload values. A second 15 minute period of load, 1 h following the end of the first, produced smaller increases in the levels of release that were statistically significant only for the first 0-5 minute sample during load (preload compared to load mean values, PGE, 1.09-1.66 ng/ml, p less than 0.02; 6-keto-PGF1 alpha, 401-558 pg/ml, p less than 0.04). Immunolocalization revealed PGE and 6-keto-PGF1 alpha in lining cells and 6-keto-PGF1 alpha but not PGE in osteocytes. Addition to the medium of 1 microM PGE2, approximating the concentration produced by loading, had no significant effect on the specific activity of the extractable RNA fraction labeled with [3H]uridine, whereas 1 microM PGI2 produced an increase similar to that seen previously with loading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomechanical competence of iliac crest trabecular bone in autosomal dominant osteopetrosis type I

Cylindrical horizontal iliac crest trabecular bone biopsies were obtained from 9 patients with autosomal dominant osteopetrosis type I and 18 normal controls of comparable age/sex match. Maximum compressive stress, maximum stiffness, energy absorption capacity and maximum strain were calculated from load-deformation curves after a compression test. Ash density of the bone samples was measured after incineration. The maximum compressive stress was significantly increased in the patient group (12.6 +/- 2.6 (SE) MPa vs. 3.3 +/- 0.4 MPa, p less than 0.01), as was the ash density (0.61 +/- 0.05 g/cm3 versus 0.27 +/- 0.02 g/cm3, p less than 0.01). After correction for ash density (normalized maximum stress) the strength of the trabecular bone samples was still significantly increased in the patients (19.7 +/- 6.4 MPa x cm3/g versus 12.0 +/- 1.2 MPa x cm3/g, p less than 0.01). The maximum stiffness and energy absorption capacity were higher in the patients (p less than 0.01), with a corresponding lower maximum strain value (p less than 0.05). The maximum compressive stress correlated closely to the maximum stiffness and energy absorption capacity in both patients and controls, whereas no correlation to maximum strain was found. The maximum compressive stress thus seems to be representative for the two other biomechanical parameters. No significant correlations between age and maximum compressive stress (R = 0.38), ash density (R = 0.08), or normalized maximum stress (R = 0.45) were observed in type I osteopetrosis, whereas significant age-dependent decreases in maximum compressive stress (R = -0.65, p less than 0.02) and in ash density (R = -0.57, p less than 0.02) were observed in normal individuals.(ABSTRACT TRUNCATED AT 250 WORDS)