Effects of vitamin K2 administration on calcium balance and bone mass in young rats fed normal or low calcium diet

OBJECTIVE: The purpose of this study was to examine the effects of vitamin K2 administration on calcium balance and bone mass in young rats fed a normal or low calcium diet. METHODS: Forty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into four groups with 10 rats in each group: 0.5% (normal) calcium diet, 0.1% (low) calcium diet, 0.5% calcium diet + vitamin K2 (menatetrenone, 30 mg/100 g chow diet), and 0.1% calcium diet + vitamin K2. After 10 weeks of feeding, serum calcium and calciotropic hormone levels were measured, and intestinal calcium absorption and renal calcium reabsorption were evaluated. Bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. RESULTS: Feeding a low calcium diet induced hypocalcemia, increased serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels with decreased serum 25-hydrovyvitamin D [25(OH)D] level, stimulated intestinal calcium absorption and renal calcium reabsorption, and reduced cortical bone mass as a result of decreased periosteal bone gain and enlarged marrow cavity, but did not significantly influence cancellous bone mass. Vitamin K2 administration in rats fed a low calcium diet stimulated renal calcium reabsorption, retarded the abnormal elevation of serum PTH level, increased cancellous bone mass, and retarded cortical bone loss, while vitamin K2 administration in rats fed a normal calcium diet stimulated intestinal calcium absorption by increasing serum 1,25(OH)2D level, and increased cortical bone mass. CONCLUSION: This study clearly shows the differential response of calcium balance and bone mass to vitamin K2 administration in rats fed a normal or low calcium diet.     

Adaptive changes in micromechanical environments of cancellous and cortical bone in response to in vivo loading and disuse

The skeleton accommodates changes in mechanical environments by increasing bone mass under increased loads and decreasing bone mass under disuse. However, little is known about the adaptive changes in micromechanical behavior of cancellous and cortical tissues resulting from loading or disuse. To address this issue, in vivo tibial loading and hindlimb unloading experiments were conducted on 16-week-old female C57BL/6J mice. Changes in bone mass and tissue-level strains in the metaphyseal cancellous and midshaft cortical bone of the tibiae, resulting from loading or unloading, were determined using microCT and finite element (FE) analysis, respectively. We found that loading- and unloading-induced changes in bone mass were more pronounced in the cancellous than cortical bone. Simulated FE-loading showed that a greater proportion of elements experienced relatively lower longitudinal strains following load-induced bone adaptation, while the opposite was true in the disuse model. While the magnitudes of maximum or minimum principal strains in the metaphyseal cancellous and midshaft cortical bone were not affected by loading, strains oriented with the long axis were reduced in the load-adapted tibia suggesting that loading-induced micromechanical benefits were aligned primarily in the loading direction. Regression analyses demonstrated that bone mass was a good predictor of bone tissue strains for the cortical bone but not for the cancellous bone, which has complex microarchitecture and spatially-variant strain environments. In summary, loading-induced micromechanical benefits for cancellous and cortical tissues are received primarily in the direction of force application and cancellous bone mass may not be related to the micromechanics of cancellous bone.     

Wnt16 Is Associated with Age-Related Bone Loss and Estrogen Withdrawal in Murine Bone

Genome Wide Association Studies suggest that Wnt16 is an important contributor to the mechanisms controlling bone mineral density, cortical thickness, bone strength and ultimately fracture risk. Wnt16 acts on osteoblasts and osteoclasts and, in cortical bone, is predominantly derived from osteoblasts. This led us to hypothesize that low bone mass would be associated with low levels of Wnt16 expression and that Wnt16 expression would be increased by anabolic factors, including mechanical loading. We therefore investigated Wnt16 expression in the context of ageing, mechanical loading and unloading, estrogen deficiency and replacement, and estrogen receptor α (ERα) depletion. Quantitative real time PCR showed that Wnt16 mRNA expression was lower in cortical bone and marrow of aged compared to young female mice. Neither increased nor decreased (by disuse) mechanical loading altered Wnt16 expression in young female mice, although Wnt16 expression was decreased following ovariectomy. Both 17β-estradiol and the Selective Estrogen Receptor Modulator Tamoxifen increased Wnt16 expression relative to ovariectomy. Wnt16 and ERβ expression were increased in female ERα^-/- mice when compared to Wild Type. We also addressed potential effects of gender on Wnt16 expression and while the expression was lower in the cortical bone of aged males as in females, it was higher in male bone marrow of aged mice compared to young. In the kidney, which we used as a non-bone reference tissue, Wnt16 expression was unaffected by age in either males or females. In summary, age, and its associated bone loss, is associated with low levels of Wnt16 expression whereas bone loss associated with disuse has no effect on Wnt16 expression. In the artificially loaded mouse tibia we observed no loading-related up-regulation of Wnt16 expression but provide evidence that its expression is influenced by estrogen receptor signaling. These findings suggest that while Wnt16 is not an obligatory contributor to regulation of bone mass per se, it potentially plays a role in influencing pathways associated with regulation of bone mass during ageing and estrogen withdrawal.     

Effects of Multi-Deficiencies-Diet on Bone Parameters of Peripheral Bone in Ovariectomized Mature Rat

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.     

Mechanical Loading Synergistically Increases Trabecular Bone Volume and Improves Mechanical Properties in the Mouse when BMP Signaling Is Specifically Ablated in Osteoblasts

Bone homeostasis is affected by several factors, particularly mechanical loading and growth factor signaling pathways. There is overwhelming evidence to validate the importance of these signaling pathways, however, whether these signals work synergistically or independently to contribute to proper bone maintenance is poorly understood. Weight-bearing exercise increases mechanical load on the skeletal system and can improves bone quality. We previously reported that conditional knockout (cKO) of Bmpr1a, which encodes one of the type 1 receptors for Bone Morphogenetic Proteins (BMPs), in an osteoblast-specific manner increased trabecular bone mass by suppressing osteoclastogenesis. The cKO bones also showed increased cortical porosity, which is expected to impair bone mechanical properties. Here, we evaluated the impact of weight-bearing exercise on the cKO bone phenotype to understand interactions between mechanical loading and BMP signaling through BMPR1A. Male mice with disruption of Bmpr1a induced at 9 weeks of age, exercised 5 days per week on a motor-driven treadmill from 11 to 16 weeks of age. Trabecular bone volume in cKO tibia was further increased by exercise, whereas exercise did not affect the trabecular bone in the control genotype group. This finding was supported by decreased levels of osteoclasts in the cKO tibiae. The cortical porosity in the cKO bones showed a marginally significant decrease with exercise and approached normal levels. Exercise increased ductility and toughness in the cKO bones. Taken together, reduction in BMPR1A signaling may sensitize osteoblasts for mechanical loading to improve bone mechanical properties.     

Joint moment contributions to swing knee extension acceleration during gait in children with spastic hemiplegic cerebral palsy

Inadequate peak knee extension during the swing phase of gait is a major deficit in individuals with spastic cerebral palsy (CP). The biomechanical mechanisms responsible for knee extension have not been thoroughly examined in CP. The purpose of this study was to assess the contributions of joint moments and gravity to knee extension acceleration during swing in children with spastic hemiplegic CP. Six children with spastic hemiplegic CP were recruited (age=13.4±4.8 years). Gait data were collected using an eight-camera system. Induced acceleration analysis was performed for each limb during swing. Average joint moment and gravity contributions to swing knee extension acceleration were calculated. Total swing and stance joint moment contributions were compared between the hemiplegic and non-hemiplegic limbs using paired t-tests (p<0.05). Swing limb joint moment contributions from the hemiplegic limb decelerated swing knee extension significantly more than those of the non-hemiplegic limb and resulted in significantly reduced knee extension acceleration. Total stance limb joint moment contributions were not statistically different. Swing limb joint moment contributions that decelerated knee extension appeared to be the primary cause of inadequate knee extension acceleration during swing. Stance limb muscle strength did not appear to be the limiting factor in achieving adequate knee extension in children with CP. Recent research has shown that the ability to extend the knee during swing is dependent on the selective voluntary motor control of the limb. Data from individual participants support this concept.     

Cortical bone thickness can adapt locally to muscular loading while changing with age

Mechanical loading of muscle action is concentrated at muscle attachment sites; thus there may be a potential for site-specific variation in cortical bone thickness. Humeri from an early 20th-century Finnish (Helsinki) and two medieval English (Newcastle, Blackgate and York, Barbican) populations were subjected to pQCT scanning to calculate site-specific cross-sectional cortical bone area (CA) for four locations and to measure cortical thickness at muscle attachment sites and non-attachment sites. We found that CA at 80% of humerus length was significantly reduced compared to more distal cross-sections, which can be due to reduced stresses at the proximal shaft. The principal direction of loading at 80% humerus length was towards mediolateral plane, likely due to fixing the humerus close to the torso. At 35% the main direction of loading was towards anteroposterior plane, reflecting elbow flexing forces. The principal direction of loading varied between populations, sides and sexes at 50% humerus length due to preference between elbow and shoulder joint; thus this location might be useful when trying to infer differences in activity. These changes are likely due to overall shaft adaptation to forces acting at the humerus. In addition, we found a potential for site-specific variation in cortical thickness; cortical bone at muscle attachment sites was significantly thicker compared to non-attachment sites. Lastly, CA at 35% of humerus length and cortical thickness at non-attachment sites decreased with age. These results underline the importance of muscle loading for bone mass preservation as well as indicate that a site-specific variation of bone mass is possible.     

Site specific bone adaptation response to mechanical loading

Over 25 million Americans suffer from osteoporosis. Bone size and strength depends both upon the level of adaptation due to physical activity (applied load), and genetics. We hypothesized that bone adaptation to loads differs among mice breeds and bone sites. Forty-five adult female mice from three inbred strains (C57BL/6 [B6], C3H/HeJ [C3], and DBA/2J [D2]) were loaded at the right tibia and ulna in vivo with non-invasive loading devices. Each loading session consisted of 99 cycles at a force range that induced approximately 2000 microstrain (microepsilon) at the mid-shaft of the tibia (2.5 to 3.5 N force) and ulna (1.5 to 2 N force). The right and left ulnae and tibiae were collected and processed using protocols for histological undecalcified cortical bone slides. Standard histomorphometry techniques were used to quantify new bone formation. The histomorphometric variables include percentage mineralizing surface (%MS), mineral apposition rate (MAR), and bone formation rate (BFR). Net loading response [right-left limb] was compared between different breeds at tibial and ulnar sites using two-way ANOVA with repeated measures (p<0.05). Significant site differences in bone adaptation response were present within each breed (p<0.005). In all the three breeds, the tibiae showed greater percentage MS, MAR and BFR than the ulna at similar in vivo load or mechanical stimulus (strain). These data suggest that the bone formation due to loading is greater in the tibiae than the ulnae. Although, no significant breed-related differences were found in response to loading, the data show greater trends in tibial bone response in B6 mice as compared to D2 and C3 mice. Our data indicate that there are site-specific skeletal differences in bone adaptation response to similar mechanical stimulus.     

Disuse in adult male rats attenuates the bone anabolic response to a therapeutic dose of parathyroid hormone.

Intermittent treatment with parathyroid hormone (PTH) increases bone formation and prevents bone loss in hindlimb-unloaded (HLU) rats. However, the mechanisms of action of PTH are incompletely known. To explore possible interactions between weight bearing and PTH, we treated 6-mo-old weight-bearing and HLU rats with a human therapeutic dose (1 microg.kg(-1).day(-1)) of human PTH(1-34) (hPTH). Cortical and cancellous bone formation was measured in tibia at the diaphysis proximal to the tibia-fibula synostosis and at the proximal metaphysis, respectively. Two weeks of hindlimb unloading resulted in a dramatic decrease in the rate of bone formation at both skeletal sites, which was prevented by PTH treatment at the cancellous site only. In contrast, PTH treatment increased cortical as well as cancellous bone formation in weight-bearing rats. Two-way ANOVA revealed that hPTH and HLU had independent and opposite effects on all histomorphometric indexes of bone formation [mineral apposition rate (MAR), double-labeled perimeter (dLPm), and bone formation rate (BFR)] at both skeletal sites. The bone anabolic effects of weight bearing and hPTH on dLPm and BFR at the cortical site were additive, as were the effects on MAR at the cancellous site. In contrast, weight bearing and hPTH resulted in synergistic increases in cortical bone MAR and cancellous bone dLPm and BFR. We conclude that weight bearing and PTH act cooperatively to increase bone formation by resulting in site-specific additive and synergistic increases in indexes of osteoblast number and activity, suggesting that weight-bearing exercise targeted to osteopenic skeletal sites may improve the efficacy of PTH therapy for osteoporosis.     

Muscle-bone relationships in the lower leg of healthy pre-pubertal females and males

Muscle-bone relationships in healthy pre-pubertal children were investigated using four muscle measures as predictors of tibial strength: 66% tibia cross-sectional muscle area (CSMA) by pQCT; leg lean mass (LLM) by DXA; and muscle power (Power) and force (Force) measured during a two-footed jump. Polar strength strain index (pSSI), a calculated surrogate for bone strength at the 20% distal tibia, was obtained on 105 (54 male) self-assessed pre-pubertal children. The amount of muscle (CSMA, LLM) may influence bone strength more than muscle strength (Power, Force) during periods of rapid growth. Correlations and multiple regression partial-R values from models controlling for age, sex, height and weight were obtained for each muscle predictor. CSMA, LLM, Power and Force were positively correlated with pSSI (R=0.84, 0.92, 0.85; 0.66, respectively, all p<0.01). Partial-R values were highest for LLM (partial-R=0.21), similar for CSMA and Power (0.14, 0.15, respectively) and lowest for Force (0.04) in predicting pSSI. Muscle predictors were associated with total and cortical area (R=0.59 to 0.90; p<0.01 for all), but not cortical vBMD at the 20% distal tibia site. These data support relationships between muscle predictors and bone parameters measured by pQCT in healthy pre-pubertal children.     

Osteosclerosis in F344/DuCrj rats

Osteosclerosis was observed in the tibia and sternum in F344/DuCrj rats of both sexes at 6, 18 and 30 months of age. The lesion first seen was a proliferation of osteogenic tissues on the marrow surface of the cortical bone and bone trabeculae, resulting in replacement of the marrow cavity by lamellar bone. Most of the affected rats had associated degenerative osteoarthrosis and regressive changes of the growth plate. Osteosclerosis was considered to be an aging change, lesions were observed at 6 months and increased in frequency with age.     

Additive effect of vitamin K2 and risedronate on long bone mass in hypophysectomized young rats.

Hypophysectomy (HX) arrests bone growth and induces osteopenia in the long bones of rats. The present study investigated the combined effect of vitamin K(2) and risedronate on long bone mass in HX rats, in order to determine whether treatment with these two agents had an additive effect. Forty female Sprague-Dawley rats were hypophysectomized at 6 weeks of age by the supplier, and were shipped to our laboratory at three days after surgery along with ten intact rats that served as age-matched controls. The study was started on the day when the rats were received. Three HX rats were excluded from the study because of the failure of HX. Forty-seven rats (6 weeks old) were assigned to the following 5 groups by the stratified weight randomization method: intact controls, HX alone, HX + vitamin K(2) (30 mg/kg, p.o., daily), HX + risedronate (2.5 microg/kg, s.c., 5 days a week), and HX + vitamin K(2) + risedronate. The dosing period was 4 weeks. HX resulted in a decrease of the femoral bone area, bone mineral content (BMC) and bone mineral density (BMD), as well as a decrease in the cancellous bone mass of the proximal tibial metaphysis and the total tissue and cortical areas of the tibial diaphysis. These changes were associated with a marked reduction in the serum level of insulin like growth factor (IGF)-I and with elevation of serum alkaline phosphatase (ALP) and pyridinoline. Administration of vitamin K(2) increased the serum ALP level in HX rats, but did not affect any of the other parameters. On the other hand, risedronate ameliorated the decrease of femoral BMD and cancellous bone mass at the proximal tibial metaphysis in HX rats without affecting the serum IGF-I level, as a result of not causing a significant elevation of serum pyridinoline. Vitamin K(2) and risedronate combined had an additive effect on the femoral bone area, BMC and BMD, and the combined treatment group did not show any significant reduction of the total tissue and cortical areas at the tibial diaphysis, as well as a reduced serum pyridinoline level compared with untreated rats and an increased serum ALP level compared with untreated or risedronate-treated rats. These results suggest that risedronate had a positive effect on the BMD and cancellous bone mass of long bones in HX rats. Despite the lack of a significant effect of vitamin K(2) on bone mass parameters, it had an additive effect with risedronate on the BMC, BMD and cortical bone mass of long bones in HX rats.     

Hindlimb unloading has a greater effect on cortical compared with cancellous bone in mature female rats.

This study was designed to determine the effects of 28 days of hindlimb unloading (HU) on the mature female rat skeleton. In vivo proximal tibia bone mineral density and geometry of HU and cage control (CC) rats were measured with peripheral quantitative computed tomography (pQCT) on days 0 and 28. Postmortem pQCT, histomorphometry, and mechanical testing were performed on tibiae and femora. After 28 days, HU animals had significantly higher daily food consumption (+39%) and lower serum estradiol levels (-49%, P = 0.079) compared with CC. Proximal tibia bone mineral content and cortical bone area significantly declined over 28 days in HU animals (-4.0 and 4.8%, respectively), whereas total and cancellous bone mineral densities were unchanged. HU animals had lower cortical bone formation rates and mineralizing surface at tibial midshaft, whereas differences in similar properties were not detected in cancellous bone of the distal femur. These results suggest that cortical bone, rather than cancellous bone, is more prominently affected by unloading in skeletally mature retired breeder female rats.     

Structural and Mechanical Improvements to Bone Are Strain Dependent with Axial Compression of the Tibia in Female C57BL/6 Mice

Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function.     

Effects of vitamin D supplementation on calcium balance and bone growth in young rats fed normal or low calcium diet

OBJECTIVE: We examined the effect of vitamin D supplementation on bone growth in young rats fed a normal or low calcium diet. METHODS: Fifty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into five groups with 10 rats in each group: baseline control, 0.5% (normal) or 0.1% (low) calcium diet, and 0.5 or 0.1% calcium diet + vitamin D (25 microg/100 g, food intake). Duration of the experiment was 10 weeks. RESULTS: Vitamin D supplementation stimulated intestinal calcium absorption and increased urinary calcium excretion in rats fed a low or normal calcium diet. Vitamin D supplementation prevented the reduction in periosteal bone gain but enhanced enlargement of the marrow cavity and reduced the maturation-related cancellous bone gain in rats fed a low calcium diet, and increased the maturation-related cancellous and cortical bone gains in rats fed a normal calcium diet. CONCLUSION: This study shows the differential effects of vitamin D supplementation on born growth in young rats fed a normal or low calcium diet.     

Indicators of erythrocyte formation and degradation in rats with either vitamin A or iron deficiency

Vitamin A deficiency produces anemia and altered iron status. In this study with rats we tested two hypotheses regarding vitamin A deficiency: (1) that it impairs erythropoiesis, leading to an increased red cell turnover, and (2) that it inhibits the glycosylation of transferrin. Erythropoietic activity was assessed indirectly by determining the myeloid:erythroid ratio in bone marrow smears, the number of erythroid colonies in the red pulp of spleen, the blood reticulocyte index, and zinc protoporphyrin and plasma transferrin receptor concentrations. Transferrin glycosylation was assessed by measuring the sialic acid content of transferrin. The effects of vitamin A deficiency were compared with those of iron deficiency. Iron deficiency produced anemia and low iron levels in organs. Vitamin A deficiency produced low levels of plasma and hepatic retinol, and it induced decreased plasma total iron-binding capacity and raised iron levels in tibia and spleen. Short- but not long-term iron deficiency reduced the number of erythroid colonies in spleen; vitamin A deficiency had no influence. Neither iron nor vitamin A deficiency influenced the myeloid:erythroid ratio in bone marrow smears and the blood reticulocyte production. Plasma transferrin receptor and erythrocyte zinc protoporphyrin concentrations were not affected by vitamin A deficiency but increased with iron deficiency. Vitamin A deficiency did not stimulate erythrocyte breakdown, as indicated by unaltered plasma lactate dehydrogenase activity and reduced plasma total bilirubin levels. Both vitamin A and iron deficiencies raised the proportion of multiple sialylated transferrins in plasma. Thus, we have not found evidence that vitamin A deficiency affects erythropoiesis and erythrocyte turnover. The iron accumulation in spleen and bone marrow may be related to reduced iron transport due to inhibition of transferrin synthesis rather than inhibition of transferrin sialylation.     

Using digital image correlation to determine bone surface strains during loading and after adaptation of the mouse tibia

Previous models of cortical bone adaptation, in which loading is imposed on the bone, have estimated the strains in the tissue using strain gauges, analytical beam theory, or finite element analysis. We used digital image correlation (DIC), tracing a speckle pattern on the surface of the bone during loading, to determine surface strains in a murine tibia during compressive loading through the knee joint. We examined whether these surface strains in the mouse tibia are modified following two weeks of load-induced adaptation by comparison with contralateral controls. Results indicated non-uniform strain patterns with isolated areas of high strain (0.5%), particularly on the medial side. Strain measurements were reproducible (standard deviation of the error 0.03%), similar between specimens, and in agreement with strain gauge measurements (between 0.1 and 0.2% strain). After structural adaptation, strains were more uniform across the tibial surface, particularly on the medial side where peak strains were reduced from 0.5% to 0.3%. Because DIC determines local strains over the entire surface, it will provide a better understanding of how strain stimulus influences the bone response during adaptation.     

Bone disease in children with homozygous β-thalassemia

The histological features of thalassemic bone are imperfectly known, and the roles of bone marrow hyperactivity, iron overload or vitamin D deficiency in the pathogenesis of the disease are not clearly identified. In this study we examined iliac crest biopsies from 17 transfusion-dependent children with homozygous β-thalassemia and severe radiological skeletal thalassemic changes, including widening of medullary spaces and osteoporosis. Rachitic lesions were not observed. Serum ferritin concentrations were increased in all but one subject. Iron deposits were histochemically detected in bone marrow, at the marrow-bone interface, along cement lines and mineralizing perimeters. Minor changes were present in trabecular bone, and osteomalacia was absent. By contrast, cortical bone exhibited severe changes including fissures and focal mineralization defects. Plasma 25-hydroxyvitamin D (25(OH)D) concentrations measured during the winter (December–May, 6.5 ± 4.9 ng/ml, mean ± SD, n = 6) and during the summer (June–November, 13.8 ± 8.4 ng/ml, n = 9) did not differ from those of age-matched children living in the same country. Seven patients had moderate hypocalcemia but no biological signs suggestive of vitamin D deficiency: all had normal alkaline phosphatase activity, normal or slightly elevated plasma phosphate, only two had low plasma 25(OH)D concentrations and two others supranormal values of plasma immunoreactive parathyroid hormone.These results show that iron overload and vitamin D deficiency do not seem to play an important role in the pathogenesis of thalassemic bone disease, which is characterized by cortical lesions probably related to marrow hyperactivity.     

Characterizing gait induced normal strains in a murine tibia cortical bone defect model

The critical role that mechanical stimuli serve in mediating bone repair is recognized but incompletely understood. Further, previous attempts to understand this role have utilized application of externally applied mechanical loads to study the tissue’s response. In this project, we have therefore endeavored to capitalize on bone’s own consistently diverse loading environment to develop a novel model that would enable assessment of the influence of physiologically engendered mechanical stimuli on cortical defect repair. We used an inverse dynamics approach with finite element analysis (FEA) to first quantify normal strain distributions generated in mouse tibia during locomotion. The strain environment of the tibia, as previously reported for other long bones, was found to arise primarily due to bending and was consistent in orientation through the stance phase of gait. Based on these data, we identified three regions within a transverse cross-section of the mid-diaphysis as uniform locations of either peak tension, peak compression, or the neutral axis of bending (i.e. minimal strain magnitude). We then used FEA to quantify the altered strain environment that would be produced by a 0.6 mm diameter cylindrical cortical bone defect at each diaphyseal site and, in an in situ study confirmed our ability to accurately place defects at the desired diaphyseal locations. The resulting model will enable the exploration of cortical bone healing within the context of physiologically engendered mechanical strain.     

Trabecular Bone Adaptation to Low-Magnitude High-Frequency Loading in Microgravity

Exposure to microgravity causes loss of lower body bone mass in some astronauts. Low-magnitude high-frequency loading can stimulate bone formation on earth. Here we hypothesized that low-magnitude high-frequency loading will also stimulate bone formation under microgravity conditions. Two groups of six bovine cancellous bone explants were cultured at microgravity on a Russian Foton-M3 spacecraft and were either loaded dynamically using a sinusoidal curve or experienced only a static load. Comparable reference groups were investigated at normal gravity. Bone structure was assessed by histology, and mechanical competence was quantified using μCT and FE modelling; bone remodelling was assessed by fluorescent labelling and secreted bone turnover markers. Statistical analyses on morphometric parameters and apparent stiffness did not reveal significant differences between the treatment groups. The release of bone formation marker from the groups cultured at normal gravity increased significantly from the first to the second week of the experiment by 90.4% and 82.5% in response to static and dynamic loading, respectively. Bone resorption markers decreased significantly for the groups cultured at microgravity by 7.5% and 8.0% in response to static and dynamic loading, respectively. We found low strain magnitudes to drive bone turnover when applied at high frequency, and this to be valid at normal as well as at microgravity. In conclusion, we found the effect of mechanical loading on trabecular bone to be regulated mainly by an increase of bone formation at normal gravity and by a decrease in bone resorption at microgravity. Additional studies with extended experimental time and increased samples number appear necessary for a further understanding of the anabolic potential of dynamic loading on bone quality and mechanical competence.