The effect of maternal HMB supplementation on bone mechanical and geometrical properties, as well as histomorphometry and immunolocalization of VEGF, TIMP2, MMP13, BMP2 in the bone and cartilage tissue of the humerus of their newborn piglets.

The presented experiment focuses on assessing the impact of HMB (hydroxy-ß-methobutyrate) supplementation of mothers during pregnancy on the development of the skeletal system of their offspring. For this purpose, an experiment was carried out on 12 clinically healthy sows of the Great White Poland breed, which were divided randomly into two groups the control and the HMB group. All animals were kept under standard conditions and received the same feed for pregnant females. In contrast, females from the HMB group between 70 and 90 days were supplemented with 3-hydroxy-3-methylbutyle in the amount of 0.2g/kg b.w/day. Immediately after birth, the piglets were also divided into groups based on: sex, and presence or lack HMB supplementation, and subsequently were euthanized and humerus bones from all piglets were collected. Mother's HMB supplementation during pregnancy affected the multiple index of their offspring. The higher humerus mass and length was observed with the greater effect in males. Maternal supplementation also influenced on the geometrical and mechanical properties of the humerus as in the case of mass, this effect was higher in males. Also, the collagen structure of the compacted and trabecular bone changed under the HMB addition. Maternal supplementation also affected the expression of selected proteins in growth cartilage and trabecular bone. The obtained results show that the administration to the mother during pregnancy by the HMB significantly affects the development of the humerus in many ways. The obtained results also confirm the utility of such experiments in understanding of the importance of the pregnancy diet as an develop and adaptable factor of offspring organisms and are the base for further research in that area as well as in the protein markers expression area.

Dietary 2-oxoglutarate prevents bone loss caused by neonatal treatment with maximal dexamethasone dose.

Synthetic glucocorticoids (GCs) are widely used in the variety of dosages for treatment of premature infants with chronic lung disease, respiratory distress syndrome, allergies, asthma, and other inflammatory and autoimmune conditions. Yet, adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Conversely, 2-oxoglutarate (2-Ox), a precursor of glutamine, glutamate, and collagen amino acids, exerts protective effects on bone development. Our aim was to elucidate the effect of dietary administered 2-Ox on bone loss caused by neonatal treatment with clinically relevant maximal therapeutic dexamethasone (Dex) dose. Long bones of neonatal female piglets receiving Dex, Dex+2-Ox, or untreated were examined through measurements of mechanical properties, density, mineralization, geometry, histomorphometry, and histology. Selected hormones, bone turnover, and growth markers were also analyzed. Neonatal administration of clinically relevant maximal dose of Dex alone led to over 30% decrease in bone mass and the ultimate strength ( P < 0.001 for all). The length (13 and 7% for femur and humerus, respectively) and other geometrical parameters (13-45%) decreased compared to the control ( P < 0.001 for all). Dex impaired bone growth and caused hormonal imbalance. Dietary 2-Ox prevented Dex influence and vast majority of assessed bone parameters were restored almost to the control level. Piglets receiving 2-Ox had heavier, denser, and stronger bones; higher levels of growth hormone and osteocalcin concentration; and preserved microarchitecture of trabecular bone compared to the Dex group. 2-Ox administered postnatally had a potential to maintain bone structure of animals simultaneously treated with maximal therapeutic doses of Dex, which, in our opinion, may open up a new opportunity in developing combined treatment for children treated with GCs. Impact statement The present study has showed, for the first time, that dietary 2-oxoglutarate (2-Ox) administered postnatally has a potential to improve/maintain bone structure of animals simultaneously treated with maximal therapeutic doses of dexamethasone (Dex). It may open the new direction in searching and developing combined treatment for children treated with glucocorticoids (GCs) since growing group of children is exposed to synthetic GCs and adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Currently proposed combined therapies have numerous side effects. Thus, this study proposed a new direction in combined therapies utilizing dietary supplementation with glutamine derivative. Impairment caused by Dex in presented long bones animal model was prevented by dietary supplementation with 2-Ox and vast majority of assessed bone parameters were restored almost to the control level. These results support previous thesis on the regulatory mechanism of nutrient utilization regulated by glutamine derivatives and enrich the nutritional science.

Lycopene treatment against loss of bone mass, microarchitecture and strength in relation to regulatory mechanisms in a postmenopausal osteoporosis model.

Lycopene supplementation decreases oxidative stress and exhibits beneficial effects on bone health, but the mechanisms through which it alters bone metabolism in vivo remain unclear. The present study aims to evaluate the effects of lycopene treatment on postmenopausal osteoporosis. Six-month-old female Wistar rats (n=264) were sham-operated (SHAM) or ovariectomized (OVX). The SHAM group received oral vehicle only and the OVX rats were randomized into five groups receiving oral daily lycopene treatment (mg/kg body weight per day): 0 OVX (control), 15 OVX, 30 OVX, and 45 OVX, and one group receiving alendronate (ALN) (2µg/kg body weight per day), for 12weeks. Bone densitometry measurements, bone turnover markers, biomechanical testing, and histomorphometric analysis were conducted. Micro computed tomography was also used to evaluate changes in microarchitecture. Lycopene treatment suppressed the OVX-induced increase in bone turnover, as indicated by changes in biomarkers of bone metabolism: serum osteocalcin (s-OC), serum N-terminal propeptide of type 1 collagen (s-PINP), serum crosslinked carboxyterminal telopeptides (s-CTX-1), and urinary deoxypyridinoline (u-DPD). Significant improvement in OVX-induced loss of bone mass, bone strength, and microarchitectural deterioration was observed in lycopene-treated OVX animals. These effects were observed mainly at sites rich in trabecular bone, with less effect in cortical bone. Lycopene treatment down-regulated osteoclast differentiation concurrent with up-regulating osteoblast together with glutathione peroxidase (GPx) catalase (CAT) and superoxide dismutase (SOD) activities. These findings demonstrate that lycopene treatment in OVX rats primarily suppressed bone turnover to restore bone strength and microarchitecture.

[Comparison of effects of Wujia Bugu decoction) and alendronate sodium on protection the bone loss of hindlimb unloaded rats].

OBJECTIVE: To compare the effects of Wujia Bugu decoction and Alendronate sodium on protecting bone and muscle loss of hindlimb unloaded rats lasting three weeks. METHODS: March to May, 2009, 40 male Wistar rats with age of 6-week, were randomized divided to four groups (10 rats in each group): hindlimb unloaded group treated with Chinese medicine (HUC), hindlimb unloaded group treated with alendronate sodium (HUA), control group (CON), as well as hindlimb unloaded group (HU). During the experiment, rats of HUC was given Wujia Bugu decoction (including the Ciwujia, Shudihuang, Huainiuxi, Muli, etc. with the concentration of 0.704 g/ml) 10 ml/kg weight once a day, HUA was given quantitative alendronate sodium slice dissolve suspension (0.9 mg/ml) once a week. CON and HU were given double-distilled water. The experiment lasted 4 weeks,from the second to the forth week, rats in HU, HUC, HUA were hindlimb unloaded. All rats were sacrificed at the fourth weekend, the content of Ca, P and the activation of ALP in serum, Bone mineral density (BMD) of humerus and femurs, Biomechanical property of tibia and humerus, as well as the weight index of biceps and sural muscles were measured. RESULTS: Compared with CON, serum Ca of HU was significantly increased (P < 0.05), BMD, mechanical properties, muscle index of hindlimb were significantly reduce (P < 0.01), the serum Ca of HUA significantly increased (P < 0.05). Serum ALP of HUC was significantly higher than other three groups (P < 0.01). Compared with HU, femoral BMD of HUC and HUA significantly increased, tibial maximum load, maximum deflection and elastic load had increased tendency; calf muscle atrophy of HUC and HUA was alleviate 50% and 12.5% respectively (P > 0.05), humeral BMD had no significant difference, while the maximum deflection (P < 0.01) and elastic deflection (P < 0.05) in humerus of HUA were significantly lower. CONCLUSION: Herbal prescription and alendronate sodium can effectively protect the bone and muscle loss of hindlimb unloaded rats, improve its mechanical structure. Herbal prescription has advantages of relieving mechanical properties change. The effects of Wujia Bugu decoction and alendronate sodium are similar in treating space weightlessness bone loss.

Depressed bone mineralization following high dose TGF-beta1 application in an orthopedic implant model.

Several previous studies of bone repair have shown 2- to 4-fold increases in bone formation following local delivery of exogenous transforming growth factor-beta (TGF-beta). Here, we use quantitative backscatter electron microscopy to test the effect of TGF-beta1 on mineralization of regenerated bone by examining tissue samples from a previously published canine study in which we found increased bone formation. In the experiment, the proximal humeri of 10 male canines were implanted bilaterally for 28 days with porous-coated implants in the presence of a 3 mm gap between the surface of the implant and the host bone. Implants placed in the left humeri were treated with TGF-beta1 at a dose of either 120 microg (n = 5) or 335 microg (n = 5), and the implants placed in the contralateral humeri served as untreated controls. Quantitative backscatter scanning electron microscopy was used to assess the volume fraction of bone and its degree of mineralization in the 3 mm gaps. The calibrated grayscale mean and median values were depressed compared to the controls in the high dose group (p = 0.048 and p = 0.041, respectively), suggesting that high dose TGF-beta delayed or inhibited mineralization of newly formed osteoid.

The effect of calcium and vitamin D3 supplementation on the healing of the proximal humerus fracture: a randomized placebo-controlled study.

The purpose of this study was to (1) quantify the healing process of the human osteoporotic proximal humerus fracture (PHF) expressed in terms of callus formation over the fracture region using BMD scanning, and (2) quantify the impact of medical intervention with vitamin D3 and calcium on the healing process of the human osteoporotic fracture. The conservatively treated PHF was chosen in order to follow the genuine fracture healing without influence of osteosynthetic materials or casts. Thirty women (mean age = 78 years; range = 58-88) with a PHF, osteoporosis or osteopenia (based on a hip scan, WHO criteria), and not taking any drugs related to bone formation, including calcium or vitamin D supplementation, were randomly assigned to either oral 800 IU vitamin D3 plus 1 g calcium or placebo, in a double-blind prospective study. We measured biochemical, radiographic, and bone mineral density effect parameters to evaluate the impact on the healing process. Scanning procedures of the fractured shoulder included use of a fixation device to obtain the highest possible precision. Double scans of the fractured shoulder revealed a coefficient of variation (CV) on BMD measurements that improved from 2.8% immediately after fracture occurrence to 1.7% at 12 weeks (P = 0.003) approaching the 1.2% levels observed over the healthy shoulder. BMD was similar in the two groups at baseline (active 0.534 g/cm2 vs. placebo 0.518 g/cm2), and both increased over the 12-week observation period, with peak levels in week 6. By week 6 BMD levels were higher in the active group (0.623 g/cm2) compared with the placebo group (0.570 g/cm2, P = 0.006). Thirty seven percent of the patients presented with vitamin D levels below 30 nmol/l, indicative of mild vitamin D insufficiency. In conclusion, we have demonstrated that it is possible to quantify callus formation of the PHF with sufficiently high precision to demonstrate the positive influence of vitamin D3 and calcium over the first 6 weeks after fracture. Whether this results in more stable fractures, extends to other fracture types, or applies to other osteogenic bone agents such as bisphosphonates remains to be examined.

A comparison of a triple-injection axillary brachial plexus block with the humeral approach.

BACKGROUND AND OBJECTIVES: This prospective, randomized, and single-blind study compared effectiveness, performance, onset, and total anesthetic time and complications of the multiple axillary block (median, radial, and musculocutaneous nerves) with the humeral approach. METHODS: One hundred patients were randomly assigned to 2 groups. In group A (axillary) median, radial, and musculocutaneus nerves were located by a nerve stimulator and injections were made. In group H (humeral) all 4 terminal nerves of the brachial plexus were located and injections were made. A total of 40 mL mepivacaine of 1% was used. RESULTS: Complete sensory block of all 6 peripheral nerves occurred in 94% and 79% of patients in groups A and H, respectively (P < .05). The time to perform the block was shorter in group A (8 +/- 4 minutes v 11 +/- 4 minutes; P < .001); onset time was shorter in group A (16 +/- 8 minutes v 21 +/- 9 minutes; P < .05); total anesthetic time was shorter in group A (24 +/- 8 minutes v 33 +/- 10 minutes; P < .0001). Complete motor block was greater in group A (88% v 66%; P < .05). More vascular punctures occurred in group A (22% v 8%, P < .05). CONCLUSION: The triple-injection axillary block was more effective than the humeral approach as it was associated with more cases of sensory and complete motor block and gave shorter performance and onset times.

Bone tissue composition, dimensions and strength in female rats given an increased dietary level of vitamin A or exposed to 3,3',4, 4',5-pentachlorobiphenyl (PCB126) alone or in combination with vitamin C.

In previous studies we have described structural and functional changes in rat bone tissue caused by 3,3',4,4',5-pentachlorobiphenyl (PCB126). Some of the effects caused by PCB126 resemble those found in vitamin C-deficient rats, as well as those found in rats with a high dietary intake of vitamin A. The present investigation was designed to determine if these PCB126-induced changes could be inhibited by addition of vitamin C to the drinking water and if they could be evoked by vitamin A administration. Five groups of female rats were used in this study, which lasted for 12 weeks. Three of the groups were exposed to PCB126 (total dose 320 microgram/kg, bw), either alone or in combination with vitamin C added to the drinking water (1 and 10 g/l, respectively). One group was given feed with increased level of vitamin A (600000 U/kg pellet) and the fifth group served as controls. Using peripheral quantitative computed tomography (pQCT), it was found that PCB126 increased trabecular density and cortical thickness, but reduced the trabecular area. Furthermore, maximum torque and stiffness of the humerus during torsional testing and serum osteocalcin levels were reduced by PCB126. Of the PCB126 induced effects observed, addition of vitamin C only inhibited the reduction of serum osteocalcin. Like PCB126 vitamin A supplementation increased the inorganic content and the bone density and also reduced the trabecular area and polar moment of inertia but did not increase the cortical thickness or reduce maximum torque, stiffness or serum osteocalcin level. Apparently, the effects induced by PCB126 are not mediated either via decreased vitamin C level or increased vitamin A level.

Long-term excessive magnesium supplementation is deleterious whereas suboptimal supply is beneficial for bones in rats.

The long-term effects of a suboptimal magnesium supply inducing a marginal or moderate deficiency or of an excessive magnesium supplementation corresponding to a basal diet with a high pharmacological intake were investigated in 36 growing Sprague-Dawley female rats. The rats were randomly divided in three groups and received a purified diet with 7 g calcium, 5 g phosphorus and either 0.2, 0.5 or 2 g magnesium per kg diet for 7 months. At the end of the trial, plasma and erythrocyte total magnesium concentrations were significantly lower in the magnesium-deficient group than in the respective control group. Serum concentrations of 1,25-dihydroxyvitamin D, PTH and IGF-I and the length of the right humeri were not affected by the dietary treatment. The volumes corrected for body weight, the medio-lateral diameters and the ratios dry weight/length of the right humeri, and the dry weight corrected for body weight of the left tibiae and of the right humeri were significantly smaller in the magnesium-supplemented group than in the two other groups. The magnesium contents of the left tibiae and of the first lumbar vertebrae were significantly lower in the magnesium-deficient group than in the two other groups. In the right femora, dual energy X-ray absorptiometry revealed significantly smaller areas in the proximal part and significantly smaller mineral contents in the second proximal quarter in the magnesium-supplemented group compared with the two other groups. Peripheral quantitative computer tomography of the right humeri revealed in the cortex significantly larger values for the relative area, mineral content, mineral density and thickness in the magnesium-deficient group compared with the control group. The maximum point of the load-deformation curve was significantly reduced in the fifth lumbar vertebrae and in the proximal femoral metaphyses of the magnesium-supplemented group. These results indicate that the long-term suboptimal magnesium supply improved some of the parameters indicators of bone health whereas the long-term supplementation was deleterious.

Bisphosphonate effects in rat unloaded hindlimb bone loss model: three-dimensional microcomputed tomographic, histomorphometric, and densitometric analyses.

The effects of antiresorptive drugs on bone loss remain unclear. Using three-dimensional microtomography, dual X-ray/densitometry, and histomorphometry, we evaluated tiludronate effects in the bone loss model of immobilization in tail-suspended rats after 7, 13, and 23 days. Seventy-eight 12-week-old Wistar male rats were assigned to 13 groups: 1 baseline group, and for each time point, 1 control group treated with vehicle and three tail-suspended groups treated with either tiludronate (0.5 or 5 mg/kg) or vehicle, administered s. c. every other day, during the last week before sacrifice. In primary spongiosa (ISP), immobilization-induced bone loss plateaued after day 7 and was prevented by tiludronate. In secondary spongiosa (IISP), bone loss appeared at day 13 with a decrease in trabecular thickness and trabecular number (Tb.N) as assessed by three-dimensional microtomography. Osteoclastic parameters did not differ in tail-suspended rats versus control rats, whereas bone formation showed a biphasic pattern: after a marked decrease at day 7, osteoblastic activity and recruitment normalized at days 13 and 23, respectively. At day 23, the 80% decrease in bone mass was fully prevented by high-dose tiludronate with an increase in Tb.N without preventing trabecular thinning. In summary, at day 7, tiludronate prevented bone loss in ISP. After day 13, tiludronate prevented bone loss in ISP and IISP despite a further decrease in bone formation. Thus, the preventive effects of tiludronate in this model may be related to the alteration in bone modeling with an increase in Tb.N in ISP and subsequently in IISP.

Dietary xylitol supplementation prevents osteoporotic changes in streptozotocin-diabetic rats.

The effects of 10% and 20% dietary xylitol supplementation on the biomechanical properties, trabeculation, and mineral content of long bones were studied in streptozotocin-diabetic rats. Forty 3-month-old male Wistar rats were divided randomly into four groups of 10. Rats in three groups were administered a single injection of streptozotocin (50 mg/kg body weight) to induce type I diabetes, while animals in the fourth group were given a sham injection of physiological saline. The sham-injected group and one of the streptozotocin-diabetic groups were fed the basal diet, while the two diabetic groups were fed the same diet supplemented with 10% and 20% xylitol (wt/wt). After 3 months, the rats were killed and the long bones were prepared for analysis. The 10% and 20% dietary xylitol supplementation significantly prevented the type I diabetes-induced decrease in the mechanical stress resistance of the tibia in the three-point bending test, the shear stress of the femur in the torsion test, and the stress resistance of the femoral neck in the loading test. No statistically significant differences were found between any groups in the values for strain or Young's modulus in the three-point bending test, or in the values for the shear modulus of elasticity in the torsion test. These findings indicate that dietary xylitol protects against the weakening of the bone strength properties of both cortical and trabecular bone without affecting the elastic-plastic properties. Supplementation with 10% and 20% dietary xylitol significantly prevented the type I diabetes-induced decrease of humeral ash weight and tibial density. Histomorphometric data for the secondary spongiosa of the proximal tibia showed that 10% and 20% dietary xylitol supplementation also significantly prevented the type I diabetes-induced loss of trabecular bone volume. In conclusion, dietary xylitol supplementation protects against the weakening of bone biomechanical properties in streptozotocin-diabetic rats. This is related to the preserved bone mineral content and preserved trabecular bone volume.

Administration of bisphosphonate prevents disuse bone atrophy induced by tail suspension.

We recently demonstrated that osteopenia induced by rat tail-suspension was associated with an initial increase in bone resorption. To study the significance of the increase in early bone resorption for osteopenia, we investigated whether administration of YH529, a third-generation bisphosphonate, prevents the development of osteopenia as evidenced by increased wet weight of the femur, together with its calcium and phosphorus contents, when compared with those of tail-suspended rats treated with the vehicle alone. These results suggested that the initial increase in bone resorption plays an important role in the development of osteopenia induced by tail suspension.

Effects of bisphosphonate on bone metabolism in tail-suspended rats.

Our previous studies demonstrated that tail suspension causes early, transient increases in osteoclastic activity, followed by a decrease in osteoblastic activity in the hind limbs of rats. To assess whether this early increase in bone resorption is important in the development of disuse atrophy, the effect of YH529, a third generation bisphosphonate, was studied on hind limb atrophy in rats subjected to tail suspension. YH529 (YH group) or PBS (control group) were administered subcutaneously in 5-week-old male Wistar rats suspended for 7 days. In the control group, wet weight, calcium and phosphorus contents decreased significantly in the femur but they did not change in the humerus. In the YH group, however, these parameters did not change significantly in the femur, but both calcium and phosphorus increased significantly in the humerus. These results indicate that the inhibition of bone resorption by YH529 prevents the development of disuse atrophy induced by tail suspension. It is thus suggested that early increases in bone resorption are important for the development of disuse bone atrophy.

Pattern duplication by retinoic acid treatment in the regenerating limbs of Korean salamander larvae, Hynobius leechii, correlates well with the extent of dedifferentiation.

In the regenerating limbs of Korean salamanders, Hynobius leechii, retinoic acid (RA) induces duplication of skeletal structures in the proximodistal (PD) axis and often in the transverse axes. In the present study, the stage-dependent effects of RA for the duplication of limb skeletal structures at two amputation levels, the distal stylopodium and the distal zeugopodium, were studied using larval limbs of Korean salamanders. The results showed that the mean level of proximalization (MLP) by RA treatment increased during the stages of dedifferentiation and early bud formation while the MLP declined thereafter in both amputation levels. The decline of the MLP at the later stages of regeneration was due to the high frequency of hypomorphic regeneration or blocked regeneration. When the effects of RA treatment at two amputation levels were compared, the overall trends were similar but the actual timing was delayed for 2-4 days in the proximal level of amputation. Furthermore, the peak level of proximalization was achieved earlier and the peak level remained longer in the distal stylopodial level of amputation compared to the distal zeugopodial level of amputation. Since the histological observations revealed that the dedifferentiation period was also extended up to 2-4 days in the proximal level of amputation, the acid phosphatase activity during the course of regeneration was measured to look for a quantitative relationship between the enzyme activity and the states of dedifferentiation. The results show that the level and the duration of acid phosphatase activity in the upper arm regenerates are both higher and longer than those in the lower arm regenerates. Furthermore, RA treatment caused an increase in acid phosphatase activity. Thus our results suggest that the state of dedifferentiation might be closely linked to the extent of proximalization of regenerating limbs by RA treatment.

Heterogeneity of growth and turnover in the femurs and humeri of calcium-replete and -deficient C57BL/6 and SENCAR mice at sexual maturity.

The effects of dietary calcium intake on the composition and turnover of the femur and humerus were compared in two mouse strains that differ in growth kinetics and phorbol ester-induced signal transduction. C57BL/6 (control) and SENCAR (large) mice were fed calcium-deficient (0.02% Ca) or -sufficient (0.6% Ca) diets from 10 to 14 weeks of age. Bone mass was determined gravimetrically. Calcium and magnesium were determined by atomic absorption spectrophotometry, while phosphorus was determined colorimetrically. Turnover was estimated pharmacokinetically in [3H]tetracycline-labeled bone. Femur and humerus lengths, breadths, masses, and ash and mineral contents were higher in SENCAR mice than in C57BL/6 mice at 10 weeks of age and after being fed 0.02% or 0.6% calcium diets for four weeks. Relative formation was higher in C57BL/6 mice than in SENCAR mice from 10 to 14 weeks of age, resulting in greater net increases (0.6% Ca diet) or lower net decreases (0.02% Ca diet) in the calcium contents of the femurs and humeri of C57BL/6 mice, compared to SENCAR mice. Calcium-deficient feeding stimulated higher relative rates of bone resorption in both strains and affected the net changes in bone calcium contents. Thus, hereditary factors in SENCAR and C57BL/6 mice that regulate formation rates, not resorption rates or the response to dietary calcium intake, appear to modulate net changes in humerus and femur calcium contents at maturity.