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Osteopontin (OPN) and Bone Sialoprotein (BSP) are co-expressed in bone and display overlapping and complementary physiological properties. Both genes show a rapid expression response to mechanical stimulation. We used mice with single and double deletions (DKO) of BSP and OPN to assess the specificity of their roles in skeletal adaptation to loading. Two-month-old Wild-Type (WT), BSP knockout (BSP−/−), OPN−/−and DKO male mice were submitted to two mechanical stimulation regimen (n = 10 mice/group) respectively impacting trabecular bone (Hypergravity, HG) and cortical bone (Whole Body Vibration, WBV). HG increased trabecular bone volume (BV/TV) in WT femur through reduced resorption, and in BSP−/−mice femur and vertebra through increased bone formation. In contrast, HG increased the turnover of OPN−/−bone, resulting in reduced femur and vertebra BV/TV. HG did not affect DKO bones. Similarly, WBV increased cortical thickness in BSP−/−mice and decreased it in OPN−/−, without affecting structurally WT and DKO bone. Vibrated BSP−/−mice displayed increased endocortical bone formation with a drop in Sclerostin expression, and reduced periosteal osteoclasts with lower Rankl and Cathepsin K expression. In contrast, vibrated OPN−/−endocortical bone displayed decreased formation and increased osteoclast coverage. Therefore, under two regimen (HG and WBV) targeting distinct bone compartments, absence of OPN resulted in bone loss while lack of BSP induced bone gain, reflecting divergent structural adaptations. Strikingly, absence of both proteins led to a relative insensitivity to either mechanical challenge. Interplay between OPN and BSP thus appears as a key element of skeletal response to mechanical stimulation.

Background: The impaired response of glucagon-like peptide-1 (GLP-1) to meals in diabetic patients can contribute to the pathogenesis of impaired insulin secretion and post-prandial hyperglycemia. This study is aimed at the assessment of the relationship between meal-induced GLP-1 and post-prandial hyperglycemia in Type 2 diabetic patients. Methods: Twenty-one drug-naïve Type 2 diabetic patients were studied. Blood glucose and active GLP-1 levels were measured 0, 30, 60, 90, and 120 min after a standard test meal. A continuous glucose monitoring (CGM) system was applied for the following 3 days. Nutrient intake at each meal was calculated on the basis of patients’ food records. For each patient, post-prandial 120-min glucose incremental area under the curve (iAUC) was included in linear regression model exploring its relationship with total energy and carbohydrate intake, and the angular coefficient for total energy (EAC) and carbohydrate (CAC) was calculated. Results: GLP-1 levels peaked 30 min after the test meal. Logarithmically transformed 60-min GLP-1 iAUC showed a significant inverse correlation with glycated hemoglobin (HbA1c) (p<0.01). A significant inverse correlation of 60-min GLP-1 iAUC was also observed with EAC and CAC (both p<0.01), meaning that patients with a lower GLP-1 response to the test meal had a higher increment of post-prandial glucose for each additional unit of total energy or carbohydrate intake. Conclusions: In Type 2 diabetic patients, a lower GLP-1 response to meals is associated with a higher HbA1c, and with a greater degree of meal-induced hyperglycemia, both in a meal test and during CGM in “real-life” conditions.

Animal studies suggest that bone remodeling is under beta-adrenergic control via the sympathetic nervous system. The purpose of this study was to examine the preventive effect of different doses of nonspecific beta-blockers (propranolol) on trabecular and cortical bone envelopes in ovariectomized rats. Six-month-old female Wistar rats were ovariectomized (OVX, n = 60) or sham-operated (n = 15). Then, OVX rats were subcutaneously injected with 0.1 (n = 15), 5 (n = 15), or 20 (n = 15) mg/kg propranolol or vehicle (n = 15) for 10 weeks. Tibial and femoral bone mineral density (BMD) were analyzed longitudinally by dual-energy X-ray absorptiometry. At death, the left tibial metaphysis and L(4) vertebrae were removed, and microcomputed tomography (Skyscan 1072; Skyscan, Aartselaar, Belgium) was performed for trabecular bone structure investigation. Histomorphometry analysis was performed on the right proximal tibia to assess bone cell activities. After 10 weeks, OVX rats had decreased BMD and trabecular parameters and increased bone turnover, as well as cortical porosity compared with the sham group (p < 0.001). Bone architecture alteration was preserved by 0.1 mg/kg propranolol due to higher trabecular number and thickness (+50.35 and +6.81%, respectively, than OVX; p < 0.001) and lower cortical pore number (-52.38% than OVX; p < 0.001). Animals treated by 0.1 mg/kg propranolol had a lower osteoclast surface and a higher osteoblast activity compared with OVX. Animals treated by 20 mg of propranolol did not significantly differ from OVX rats. Animals treated by 5 mg of propranolol have been partially preserved from the ovariectomy. These results showed a dose effect of beta-blockers. The lower the dose of propranolol breeding, the better the preventive effect against ovariectomy.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin‐3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin‐3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin‐3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin‐3A signaling system (Semaphorin‐3A, Neuropilin‐1, and Plexins‐A1 and ‐A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin‐3A and Neuropilin‐1 are also expressed in the bone marrow. Plexin‐A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin‐A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin‐3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin‐A3/Neuropilin‐1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin‐3A. Developmental Dynamics 234:393–403, 2005. © 2005 Wiley‐Liss, Inc.

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.

Bone mineral density (BMD) was measured in 128 normal postmenopausal women at different skeletal sites: lumbar spine and proximal femur, using dual-energy X-ray absorptiometry (DXA), and the cancellous and cortical envelopes of the distal third of radius and tibia, using precise low-dose quantitative computed tomography (QCT). Multivariate analysis included chronological age, ages related to menstrual history (menopause and menarche) and anthropometric factors, e.g. height and weight, as independent predictive variables. Weight is a much-studied predictor of bone density. At sites of high bone turnover, i.e. cancellous envelope, the effect of weight appeared overshadowed by estrogen-related parameters: age-past-menopause was the first predictor of BMD in the cancellous compartment of radius and in Ward's triangle, and the number of reproductive years was the strongest predictor of BMD in the cancellous compartment of tibia and in the spine (L2–4). This suggests that in addition to menopause, the length of menstrual life should be considered as an explanation for the variations in current bone mass in postmenopausal women.

This study examined the correlations between isokinetic muscle strength of knee and elbow flexors and extensors with vertebral and femoral bone mineral density in a population of 106 women between the ages of 44 and 87 years. The absolute value of muscle strength correlated significantly with bone mineral density; muscle strength of the upper limb appeared to be more closely correlated with bone mass, while muscle strength in the lower limb was more specific for femoral mineral bone density. The most important finding that these results demonstrated was a concomitant decline in muscle strength of the upper limb and bone mineral density between the 5th and 6th decades. In contrast, they also showed a decline in muscle strength of the lower limbs after the 6th decade, occurring before the decline in bone mineral density observed between the 7th and 8th decades. From these results it would appear that other studies are required to examine the relationship between the essentially hormonal role in postmenopausal decline in muscle strength and the decline in physical activity during the senile period. These elements are important because they must be taken into account in physical exercise programmes designed to prevent osteoporosis.

Abstract: Osteoblastic cells cultured on microcarriers in bioreactors are a potentially useful tool to reproduce the in vivo three-dimensional (3D) bone network. The aim is to compare different types of 3D and two-dimensional (2D) osteoblastic culture. ROS17/2.8 cells are cultured in a bioreactor (rotating-wall vessel) or in two kinds of control (3D petri dish, 3D Percoll) and on two types of microcarrier (Cytodex 3 and Biosilon). Growth and morphology are determined by cell count and SEM, and differentiation is determined by dosage of alkaline phosphatase (ALP) activity and northern blots (ALP and osteocalcin (OC)). SEM shows that Biosilon microcarriers are the best substrate. Proliferation in the RWV and 3D petri dish is still in the exponential phase, whereas growth in the 2D culture reaches a plateau after eight days of culture. ALP activity and the ALP and OC mRNA levels are similar at day 8 for both the RWV and 3D petri dish. However, at day 10, cells are more differentiated in the RWV. The study shows that osteoblasts are both proliferate and differentiate in 3D structures. A BrDU immunocytochemical approach shows that only the cells in the periphery of the aggregates proliferate. Therefore the bioreactor may be a suitable tissue culture model for investigation of growth and differentiation processes in tissue engineering.

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.

To specify the exercise-induced changes on different skeletal sites, the effect of a 5-week endurance swin training was studied in rats. Eighteen Lyon strain (Sprague-Dawley) 5-week old female rats were divided into nine sedentary and nine swimming rats. Each swim training session was increased by 15 minutes from 2–6 hours per day. A histomorphometric study was performed at the primary and secondary spongiosa of the distal femur and at the secondary spongiosa of lumbar and thoracic vertebral bodies. After training, bone loss was observed in the secondary spongiosa of lumbar vertebral bodies (24.7%) and in the primary spongiosa of distal femur (15.2%). A tendency to bone loss was also detected in the secondary spongiosa of distal femur (10.8%), whereas no change was detected in thoracic vertebral bodies. In secondary spongiosa, bone loss was accompanied with a thinning of trabeculae. Total eroded surfaces and osteoid surfaces were significantly decreased in the three studied skeletal sites, suggesting a decreased bone turnover. The decreased thickness of osteoid seams in both lumbar vertebrae and distal femur could mean that the osteoblastic activity has also been altered at the cell level, leading to thinning of trabeculae. Five-week swim training with such duration and intensity of exercise appears unable to increase bone volume in rats and, therefore, causes adverse effects. The three studied bones seemed to adapt differently to experimental conditions. The lack of ground reaction forces induced by water immersion might have contributed to the observed bone loss. “Normal” gravity would be an important cofactor in the osteogenic effects of exercise.

Review of the literature reveals that the relationship between the extensor digitorum muscle tendon to the proximal phalanx and the metacarpophalangeal joint capsule remains unclear. The present study presents data about these relationships and consists of three parts: dissection of the region, high-gradient magnetic resonance imaging, and functional study. A total of 50 hands were used. Dissection was performed on 30, magnetic resonance studies were performed on 10, and the remaining 10 hands were used for the functional analysis. Dissection did not reveal an insertion of the extensor digitorum tendon on the base of the proximal phalanx. An extension of the dorsal part of the metacarpophalangeal joint capsule running proximally toward the palmar side of the extensor tendon was observed in eight hands. In the remaining 22 hands, only loose connective tissue was found between the articular capsule and the tendon. The development of this tissue was variable. These observations were correlated using a 7T magnetic resonance installation. The results of the functional study showed that hyperextension of the proximal phalanx increased after resection of the metacarpophalangeal structures lying under the extensor tendon. In conclusion, no real tendinous insertion of the extensor digitorum tendon on the base of the proximal phalanx could be found. Loose connective tissue was observed between the metacarpophalangeal joint capsule and the palmar aspect of the tendon, which seemed to play a secondary role in the extension of the proximal phalanx.

Microgravity-induced changes in body composition (decrease in muscle mass and increase in fat mass) and energy metabolism were studied in seven healthy male subjects during a 42-day bed-rest in a head-down tilt (HDT) position. Resting energy expenditure (REE), fat and glucose oxidation were estimated by indirect calorimetry on days 0, +8 and +40 of the HDT period. Assessments were performed both in post-absorptive conditions and following two identical test meals given at 3-h intervals. Body composition (dual x-ray absorptiometry) was measured on days 0, +27, +42. Mean post-absorptive lipid oxidation decreased from 53 (SEM 8) mg?·?min -1 (day 0) to 32 (SEM 10) mg?·?min -1 (day 8, P=0.04) and 36 (SEM 8) mg?·?min -1 (day 40, P=0.06). Mean post-absorptive glucose oxidation rose from 126 (SEM 15) mg?·?min -1 (day 0) to 164 (SEM 14) mg?·?min -1 (day 8, P=0.04) and 160 (SEM 20) mg?·?min -1 (day 40, P=0.07). Mean fat-free mass (FFM) decreased between days 0 and 42 [58.0 (SEM 1.8) kg and 55.3 (SEM 1.7) kg, P<0.01] while fat mass increased without reaching statistical significance. The mean REE decreased from 1688 (SEM 50) kcal?·?day -1 to 1589 (SEM 42) kcal?·?day -1 ( P=0.056). Changes in REE were accounted for by changes in FFM. Mean energy intake decreased from 2532 (SEM 43) kcal?· day -1 to 2237 (SEM 50) kcal?·?day -1 (day 40, P<0.01) with only a minor decrease in the proportion of fat. We concluded that changes in fat oxidation at the whole body level can be found during HDT experiments. These changes were related to the decrease in FFM and could have promoted positive fat balance hence an increase in fat mass.

After histochemical staining of tartrate‐resistant acid phosphatase (TRAP) activity, the total and active trabecular resorption surfaces and the number of osteoclasts were determined by histomorphometry on iliac crest biopsies from 36 healthy volunteers. The subjects were separated into three groups according to age and sex. Total trabecular resorption surface showed no significant variation in any group, but the fraction of active resorption surface was significantly higher in the older population. The number of TRAP cells per mm2of section area, related to trabecular bone volume or surface, showed a significant increase in elderly subjects. The mean osteoclast interface was similar in all the groups. We found a significant decrease in resorption depth between young and old populations. These results are consistent with a reduced activity of bone‐resorbing cells in advancing age. These normal values, established after histochemical identification of osteoclasts, may be applied for evaluating abnormal bone‐resorbing cell activity in metabolic bone diseases.

Without Abstract:

Our knowledge of the adaptation of human bone microgravity remains poor despite long‐term Russian spaceflights and the recent use of accurate techniques for bone mass measurements. The extent of bone deficits in the adaptation of the whole skeleton is not clear. At the tissue level, bone resorption and formation activities have been studied only in bones from rats after spaceflights lasting a few days to 3 weeks. In these animals, architectural features consistent with osteoporosis have been found in the proximal tibia. In pregnant animals the osteoclast population is increased at other skeletal sites. In areas of weight‐bearing bones that are not protected by muscular insertions, bone resorption is not markedly altered after 7 days of spaceflight and bone formation is reduced. In areas of weight‐bearing bones with muscular insertions and in non‐weight‐bearing bones, similar changes in bone cell activity are delayed. The severity of the response seems to vary with the location of the bone in the skeleton and its initial level of bone turnover. After 12.5 days the acute bone changes are less and no additional changes are observed after 21 days in space. We conclude that generalized bone deficits do not appear to be a consequence of microgravity but occur in localized areas according to the level of modeling and remodeling and of the support function of each bone at 1 g.