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The physiological role of bone vascularization in bone metabolism begins to be understood; however, its involvement in pathological situations remains poorly explored. Bone blood supply depends on both vascular density and blood flow. However, in mice, the specific evaluation of perfusion in bone suffers from a lack of easy-handling measurement tools. In the present study, we first developed a Laser Doppler Perfusion Measurement (LDPM) protocol in mouse tibia, which we validated with ex vivo and in vivo experiments. Then we carried out a study associating both structural (vascular quantitative histomorphometry) and functional (LDPM) approaches. We studied the effects of aging in 4, 7 and 17 month-old male mice and the early effects of ovariectomy in 4 month-old females. Both studies were carried out in inbred mice (C57BL/6) and in mice of mixed background (129sv/CD1). The significant differences we observed between strains in unchallenged 4 month-old animals concerned both perfusion and vascular density and depended on gender. Additionally, the age-related bone loss observed in male mice was not temporally associated with vascular changes in either strain. Between 7 and 17 months, we did not find any decrease in bone vascular density or perfusion. In contrast, ovariectomy triggered early vascular structural and functional adaptations which differed between genetic backgrounds. We observed that bone vessel density did not generally account for bone perfusion levels. In conclusion, we describe here a LDPM-based experimental protocol which provides a reproducible quantitative evaluation of bone perfusion in mouse tibia, hence allowing intergroup comparisons. This integrative structural and functional approach of bone vascularization showed that bone vascular adaptation occurs during aging or after ovariectomy and is affected by the genetic background., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Osteoporosis, or rather the localised bone loss observed in patients with spinal cord injury, as well as during any type of immobilisation involves various processes and structures including the direct response of the musculoskeletal system to unloading, the central and peripheral nervous systems and their effects on bone cells and on the vascular system, the bone remodelling unit in its marrow compartment and a number of local factors controlling cell-cell cross-talk as well as calciotropic hormones. The authors present a detailed review of these different mechanisms which are all involved regardless of the type of immobilisation: pathological, environmental, or experimental. These factors are interconnected and put bone at the centre of the regulation of body homeostasis. A better knowledge of these mechanisms should promote the development of preventive therapies for the often neglected osteoporotic fractures that occur in patients with spinal cord injury., (Copyright © 2011 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.)

Introduction: Static bone histomorphometry was applied to existing iliac bone sections originating from a 370-d 5 degrees head-down bed rest experiment. This bed rest experiment is the longest ever to have been conducted. We hypothesized that bed rest would decrease cancellous bone volume fraction and that this effect would be reversed by countermeasures., Methods: Eight healthy male subjects underwent 370 d of 5 degrees head-down bed rest. Three subjects were treated with bisphosphonate (Xidifon, potassium salt of ethane-1-hydroxy-1-disphosphonate, EHDP) combined with an exercise regimen (1-2 h x d(-1)) for the entire study period. Five subjects underwent 120 d of bed rest without countermeasures followed by 250 d of bed rest with the exercise regimen. Transiliac bone biopsies were obtained either at baseline and day 366, or at baseline, day 116, and day 366 at alternating sides of the ileum. Static histomorphometry was performed using a computerized method., Results: The 120 d of head-down bed rest without countermeasures resulted in decreased bone volume fraction BV/TV (-6.3%, p = 0.046) and trabecular number (Tb.N; -10.2%, p = 0.080) and increased trabecular separation (Tb.Sp; 14.7%, p = 0.020), whereas the 250 d of subsequent head-down bed rest with exercise treatment prevented further significant deterioration of the histomorphometric measures., Discussion: The 120 d of 5 degrees head-down bed rest without countermeasures induced significant deterioration of iliac crest cancellous bone histomorphometric properties. On average, the countermeasures consisting of either bisphosphonate and exercise, or exercise alone were able to either prevent or stop immobilization-induced changes of the iliac cancellous bone structure.

We assessed the effects of tail-suspension in two skeletal genetic backgrounds, the high C3H/HeJ (C3H) and low C57BL/6J (B6) bone masses inbred mice (male, 4-months old). Cancellous bone mass and structural parameters were evaluated in distal femoral metaphysis by three dimensional microcomputed tomography. Bone cellular activities were evaluated by histomorphometry and measurements of alkaline phosphatase activity (ALP) and osteocalcin in blood and deoxypyridinoline (D-pyr) in urine. In C3H mice, 2- and 3-week unloading experiments were performed. After an early and transient decrease in body weight, a 2-week suspension period resulted in stimulation of both bone formation rate by 45% and active osteoclastic surfaces by 19%. D-pyr did not change, but ALP and osteocalcin levels increased by 18% and 72%, respectively, in 2-week suspended mice, and osteocalcin remained elevated by 30% in the 3-week suspended mice. Such cellular modifications allowed the C3H mice to maintain their initial bone mass and trabecular structural parameters even after a 3-week suspension period. In B6 mice, 1- and 2-week unloading experiments were performed. Tail suspension resulted in decreased body weight during the first days followed by an incomplete recovery during the second week of unloading. The resorption activity was unaffected by any suspension time period, whereas a decrease of 42.5% in bone formation rate and of 21.5% in ALP were seen by the end of the first week of suspension, both values being restored after a 2-week suspension period. At this latter time, trabeculae were thinner, leading to a 24.5% cancellous bone loss. Trabecular number and connectivity, rod-plate index, and degree of anisotropy were not modified. We concluded that C3H mice constituted a unique model in which genetic background overwhelmed the usual effects of reduced biomechanical usage in bone, whereas B6 mice, compared with the standardized rat model, offered an alternative model of bone loss in a mature skeleton.

Rationale and Objectives: The ability of three-dimensional micro-computed tomography (3D-microCT) to detect changes in a rat model of disuse osteoporosis was evaluated and compared with two reference techniques: dual x-ray absorptiometry (DEXA) for bone mass, and bone histomorphometry (BHM) for bone mass and trabecular micro-architecture., Methods: Forty-two rats were divided into controls or were hindlimb unloaded for 7, 13, and 23 days. DEXA bone mineral density measurements were performed on right tibiae. Then, after plastic embedding, bone volume (BV/TV) and trabecular (Tb)-derived parameters of trabecular bone architecture (Tb Th, thickness; Tb N, number) were measured with BHM. 3D-microCT measurements of BV/TV, Tb Th, and Tb N were carried out on left tibiae., Results: Unloaded rats lost bone in a time-dependent manner. DEXA and 3D-microCT detected bone loss earlier than BHM. The decreases in Tb Th and Tb N were observed at day 13 only with 3D-microCT (P < 0.05 and P < 0.01, respectively). All bone mass and architectural parameters measured with the three techniques correlated significantly (0.59, 0.89, P < 0.001), except Tb Th., Conclusions: 3D-microCT is a valid technique for bone mass and micro-architecture measurements in this rat model of disuse osteoporosis.

Bone mass and microarchitecture are the main determinants of bone strength. Three-dimensional micro-computed tomography has the potential to examine complete bones of small laboratory animals with very high resolution in a non-invasive way. In the presented work, the proximal part of the tibiae of hindlimb unloaded and control rats were measured with 3D MicroCT, and the secondary spongiosa of the scanned region was evaluated using direct evaluation techniques that do not require model assumptions. For determination of the complete bone status, the cortex of the tibiae was evaluated and characterised by its thickness. It is shown that with the proposed anatomically conforming volume of interest (VOI), up to an eight-fold volume increase can be evaluated compared to cubic or spherical VOIs. A pronounced trabecular bone loss of -50% is seen after 23 days of tail suspension. With the new evaluation techniques, it is shown that most of this bone loss is caused by the thinning of trabeculae, and to a lesser extent by a decrease in their number. What changes most radically is the structure type: the remaining bone is more rod-like than the control group's bone. Cortical bone decreases less than trabecular bone, with only -18% after 23 days.

In an open prospective study, we evaluated differences between patients with (wRVF group) and without recurrence of vertebral fracture (woRVF group) during cyclical etidronate therapy for osteoporosis. Thirty-two patients (age 64 +/- 1.8 years) characterized by at least one osteoporotic VF were treated during 1 year. At baseline, body mass index was significantly lower (23.3 +/- 0.6 vs. 26.9 +/- 1.0 kg/m2, p< 0.05), the number of previous VFs was higher (4.0 +/- 0. 4 vs. 2.4 +/- 0.4, NS), and patients were older in the wRVF group as compared with the woRVF group (67.8 +/- 3 vs. 62.6+/- 2.2 year, NS). Trabecular bone volume (11.6 +/- 1.2 vs. 15 +/- 0.9%, p< 0.05) and trabecular number (1.06 +/- 0.08 vs. 1.27 +/- 0.05, p < 0.05) were significantly lower in the wRVF group. None of the baseline resorptive variables differed, whereas the bone formation rate (BFR) was 2-fold lower in the wRVF group (p< 0. 05). After 1 year of treatment, osteoclast number, active eroded surfaces, and resorption depth dramatically decreased in both groups (p< 0. 01). To a lesser extent, the mineral apposition rate and serum alkaline phosphatase level were significantly reduced (p< 0.05). No impaired mineralization was observed. Using X-ray microanalysis, we found no abnormality in bone mineral but a significant increase of the calcium/phosphorus ratio during treatment in the wRVF group. Our results demonstrate that recurrence of VFs within the first year of cyclical etidronate therapy was related neither to a lack of histologic response to the treatment nor induction of an abnormality of mineralization. VFs were more likely in the presence of a decreased BFR and lower trabecular connectivity, providing support for treating osteoporotic patients with etidronate early in the course of the disease.

Local immobilization is a good model for studying disuse-induced bone loss and to appreciate the effects of drugs, especially preventive action of antiresorptive therapy. In fact, increased osteoclastic activity is the main point of such a bone loss. The effect of salmon calcitonin was investigated on immobilization-induced osteoporosis in the sheep. Twenty-four nonovariectomized, adult, female, Welsh mountain sheep were submitted, by an external fixator procedure, to hock joint immobilization from the tibia to the the metatarsus for 12 weeks. The sheep were randomized into two groups receiving either an injection of placebo or salmon calcitonin (100 IU) three times per week, for 12 weeks. Histomorphometric analysis was performed on pre- and posttherapeutic transiliac bone biopsies, and on immobilized (left) and nonimmobilized calcanei removed after sacrifice. Results showed a 29% significant decrease of cancellous bone volume in the placebo group due to a significant reduced trabecular thickness when we compared immobilized versus nonimmobilized calcaneus. This structural adaptation appeared to be the consequence of an overall increased bone turnover. In the calcitonin group, same changes were observed, with a 23% reduction of bone mass in the immobilized calcaneus. By comparing calcitonin with placebo groups in both left and right calcanei, no difference was found. On the other hand, a significant increase of mineralization parameters in the iliac crest was only observed in the calcitonin group. In conclusion, salmon calcitonin, at a dose of 100 IU/day three times a week, was ineffective in preventing local disuse osteoporosis in this sheep model.

The relationship between calcitonin-induced hypocalcemia and histomorphometric parameters of bone resorption was examined in iliac crest biopsies of 30 osteoporotic patients aged 55-86 years all of whom had received a single injection of 100 UI of salmon calcitonin. Number of osteoclasts and active resorption surfaces were determined after histoenzymologic staining based on osteoclastic tartrate resistant acid phosphatase content. No significant correlation could be demonstrated between the drop of hypocalcemia and the different histomorphometric parameters. It can be concluded that the calcitonin test is useless in osteoporosis.

Acute osteoporosis is known to occur after immobilization in spinal cord injured patients and is related to an early increase in osteoclastic bone resorption. Whether osteoporosis develops in healthy immobilized human patients is still a matter of controversy. Furthermore, acute osteoporosis was thought to be a good model to study the effects of weightlessness on the human skeleton and to adapt preventive procedures. A bed rest experiment was developed in the USSR on 15 healthy human volunteers to determine the precise effects on bone structure and cell activities. A preventive protocol, including an anti-osteoclastic drug (1-hydroxyethylidene-1,1 bisphosphonic acid; K salt) was investigated. Two transiliac bone biopsies were performed on the 15 individuals before and at the end of the 120-day bed rest period. Undecalcified bone biopsies were studied with automatic and semi-automatic image analyzers specially devoted to bone histomorphometry. Trabecular bone volume, osteoid amount, and eroded surfaces were measured. Osteoclast number was measured after histochemical identification of tartrate-resistant acid phosphatase. After the bed rest period, an insignificant bone loss was observed in healthy humans while osteoclast number was highly increased. In bisphosphonate-treated subjects, osteoclast number was markedly reduced and so was osteoid amount. Bisphosphonates were shown to present a highly cytotoxic activity on osteoclasts, a finding that has never been demonstrated in normal subjects.

A histomorphometric analysis were made on iliac crest biopsies from eight healthy male volunteers submitted to a 4-month antiorthostatic bedrest. Bone mass and bone cell parameters, reflecting resorption and formation activities, were measured before and after the bedrest period. Trabecular bone volume and mean cortical thickness were not modified despite a decreased number of trabeculae and nonsignificant increase of the trabecular thickness; total and active resorption surfaces and the number of osteoclast per mm2 of trabecular surfaces do not vary significantly. Osteoid thickness does not vary but we found a reduced osteoid surface and a nonsignificant decreased osteoid volume. Our results suggest that bone architecture may be more affected by the reduction of mechanical forces than the bone mass. These modifications were supposed to be the result of an accelerated bone turnover in the early stage of immobilization. In this study, we failed to find disuse osteoporosis; however, we must point out that the new organization of the trabeculae could affect the bone mechanical properties. [ABSTRACT FROM AUTHOR]

Abstract: The preferred assessment of bone biomechanical competence in vitro is by direct mechanical testing and in vivo by X-ray absorptiometry techniques. For more than 30 years investigators have been developing alternative techniques, by ultrasonic methods, aimed at providing (i) the elastic properties in vitro without the complications related the destructive character of mechanical testing and (ii) the evaluation of the fracture risk in vivo, taking benefit of the propagation of elastic wave which are inherently affected by both material and structural bone properties. The in vitro strategy includes the use of multiscale ultrasonic assessment for the combined evaluation of both the material (or intrinsic) and the structural elastic properties in various axes of symmetry. The in vivo approach involves the use of the different propagation modes (bulk wave, guided wave, surface wave) permitted by the ultrasonic approach. We will review the in vitro and clinical data on each ultrasonic approach and discuss potential areas of development in the field of quantitative bone assessment bone. [Copyright &y& Elsevier]

We developed an automatic method to characterize mice bone architecture from three-dimensional (3D) microtomographic images. The distal metaphyses of the femur of mice were imaged using 3D synchrotron radiation microtomography at the European Synchrotron Radiation Facility (ID19) with a voxel size of 6.65 μm. Within each reconstructed volume, a region of interest was defined and trabecular and cortical bones were automatically separated. Then, 3D morphologic and topologic model-independent parameters quantifying the 3D bone architecture were computed in both regions. The technique was applied to study the response of the C57BL/6J@Ico strain of mice submitted to a model of bone loss by hind limb unloading produced by tail-suspension. [Copyright &y& Elsevier]

Background: Microgravity has been thought to induce osteoporosis because of reduced weight-bearing. However, up to now, few data have been available about its precise nature and timecourse.Methods: We measured bone mineral density (BMD) at the distal radius and tibia in 15 cosmonauts of the Russian MIR space station who sojourned in space either 1 (n=two), 2 (two), or 6 months (11). After recovery periods of similar duration to the space missions, BMD was measured for the 2-month and 6-month crews.Findings: Neither cancellous nor cortical bone of the radius was significantly changed at any of the timepoints. On the contrary, in the weight-bearing tibial site, cancellous BMD loss was already present after the first month and deteriorated with mission duration. In tibial cortices, bone loss was noted after a 2-month flight. In the 6-month group, cortical bone loss was less pronounced than that for cancellous bone. In some individuals, tibial deterioration was great. Actual BMD did not depend on preceding cumulative periods spent in space. During recovery, tibial bone loss persisted, suggesting that the time needed to recover is longer than the mission duration.Interpretation: In space, despite physical training, bone loss is an adaptive process that can become pathological after recovery on Earth. Striking interindividual variations in bone responses seem to suggest a need for adequate crew preselection. Targeted treatment or prevention strategies would be useful, not only for space purposes, but also for the increasing number of osteoporotic patients on Earth. [ABSTRACT FROM AUTHOR]