ß-Hydroxy-ß-Methylbutyrate (HMB) Supplementation Prevents Bone Loss during Pregnancy-Novel Evidence from a Spiny Mouse (Acomys cahirinus) Model.

The aim of this study was to determine the effects of ß-hydroxy-ß-methylbutyrate (HMB) supplementation during pregnancy on postpartum bone tissue quality by assessing changes in trabecular and compact bone as well as in hyaline and epiphyseal cartilage. The experiment was carried out on adult 6-month-old female spiny mice (Acomys cahirinus) divided into three groups: pregnant control (PregCont), pregnant HMB-treated (supplemented with 0.02 g/kg b.w of HMB during the second trimester of pregnancy, PregHMB), and non-pregnant females (NonPreg). Cross-sectional area and cortical index of the femoral mid-shaft, stiffness, and Young modulus were significantly greater in the PregHMB group. Whole-bone mineral density was similar in all groups, and HMB supplementation increased trabecular number. Growth plate cartilage was the thinnest, while the articular cartilage was the thickest in the PregHMB group. HMB supplementation increased the content of proteoglycans in the articular cartilage and the percentage of immature collagen content in metaphyseal trabeculae and compact bone. In summary, dietary HMB supplementation during the second trimester of pregnancy intensifies bone metabolic processes and prevents bone loss during pregnancy.

Inhibition of bone resorption blunts osteoarthritis in mice with high bone remodelling.

BACKGROUND: Osteoarthritis (OA) is characterised by cartilage degradation and bone lesions. Subchondral bone may be involved in the pathogenesis of cartilage matrix breakdown. OBJECTIVE: To assess the role of bone remodelling in OA by studying the effect of bisphosphonate on OA development in mice with high bone remodelling. METHODS: Mice overexpressing Runx2 (Runx2-Tg) under the control of collagen type I that displayed high bone remodelling were used. Joint instability was performed by partial medial meniscectomy to induce OA. RESULTS: Six weeks after surgery, tibial cartilage of Runx2-Tg mice displayed an increased number of ADAMTS-4- and ADAMTS-5-expressing chondrocytes compared with controls (p<0.05). This increase was higher in Runx2-Tg mice than in wild-type mice, although their OA score did not differ (2.5+/-0.6 vs 2.4+/-0.2, P=NS). Pamidronate reduced the OA score in Runx2-Tg mice but not in wild-type littermates (1.2+/-0.5 vs 2.7+/-0.4; p<0.05) despite the reduction of bone resorption and of the expression of cartilage proteases in both genotypes. CONCLUSIONS: These findings support the hypothesis that the level of bone resorption influences cartilage metabolism and that inhibition might prevent the progression of OA. Targeting bone resorption might therefore provide an approach to the treatment of high bone resorbing forms of OA.

Effects of PTH treatment on tibial bone of ovariectomized rats assessed by in vivo micro-CT.

UNLABELLED: Using in vivo microcomputed tomography (micro-CT), we found in parathyroid hormone (PTH)-treated osteopenic rats linear increases in cortical and trabecular, due to increased trabecular thickness and number, bone mass. Bone was formed in cavities, leading to restoral of nearly cleaved trabeculae. For the first time, effects in PTH-treated rats were analyzed longitudinally. INTRODUCTION: Our aims were to over time (1) determine changes in trabecular thickness and number after PTH, (2) compare responses to PTH between the meta- and epiphysis, (3) determine effects of PTH on mineralization and mechanical properties, (4) determine locations of new bone formation due to PTH on a microlevel, and (5) determine the predictive value of bone structural properties for gain in bone mass after PTH. METHODS: Adult rats were divided into ovariectomy (OVX; n = 8), SHAM-OVX (n = 8), and OVX and PTH treatment (n = 9). Between weeks 8 and 14, PTH rats received daily subcutaneous PTH injections (60 microg/kg/day). At weeks 0, 8, 10, 12, and 14, in vivo micro-CT scans were made of the proximal and diaphyseal tibia. After sacrifice, all tibiae were tested in three-point bending. RESULTS: PTH increased bone volume fraction linearly over time in meta- and epiphysis, accompanied by increased trabecular thickness in both and increased trabecular number only in the latter one. CT-estimated mineralization increased in trabecular and remained constant in cortical bone. Ultimate load and energy were increased and ultimate displacement and stiffness unaltered compared to SHAM rats. For those trabeculae analyzed, bone was formed initially on places where it was most beneficial for increasing their strength and later on to all surfaces.

Modulation of experimental osteoporosis in rats by the antioxidant beverage effective microorganism-X (EM-X).

Osteoporosis is a disease of aging associated with bone loss that often occurs without symptoms until microarchitectural deterioration becomes so significant that bone fracture occurs. The effective microorganism-X (EM-X) is an antioxidant beverage derived from ferment of unpolished rice, sea weeds and papaya with effective microorganisms of lactic acid bacteria, yeast and photosynthetic bacteria (containing minerals, alpha-tocopherol, lycopene, ubiquinone, saponin and flavonoids). The levels of serum estradiol (E(2)) and the bone density of the middle and epiphysis of femurs were assessed in order to determine the effect of EM-X on osteoporosis in ovariectomized rat (an animal model of postmenopausal osteoporosis). EM-X (1 ml/rat/day) was initially administrated by gavage to rats which were then allowed to consume 10% (v/v) EM-X in water freely for 3 months. There was no statistical significance of E(2) level between sham operation group and control group, indicating that sham operation did not affect E(2) level. However, the E(2) levels in the ovariectomized rats tended to increase after treatment of EM-X for 3 months. The bone density of the middle and epiphysis of femur in both sham operation and ovariectomy group decreased with time. Rats receiving EM-X for 3 months after sham operation or ovariectomy had increased bone density of the middle of femur that was statistically significant (P < 0.01 and P < 0.05). The bone density of the epiphysis of femur in both sham operation and ovariectomy group were significantly increased, an outcome highly suggestive of the beneficial effects of EM-X on bone density of the middle and the epiphysis of femur in the rats with or without ovariectomy.

Increase in bone mineral density through oral administration of shark gelatin to ovariectomized rats.

OBJECTIVE: The incidence of bovine spongiform encephalopathy has resulted in marine collagen hydrolysate (low-molecular-weight gelatin) being sold as supplements and cosmetics in Japan. Shark skin collagen is one of the important sources of marine collagen. We examined the effect of shark skin gelatin in an osteoporosis model animal. METHODS: Shark skin gelatin was orally administered to ovariectomized rats with a low-protein diet. Bone mineral density of the right femur was measured. Collagen and glycosaminoglycan in the tibial end were extracted and analyzed by western blotting and cellulose acetate membrane electrophoresis, respectively. RESULTS: Administering collagen to the ovariectomized rats resulted in the bone mineral density of the femur epiphysis being higher than that in the sham-operated rats. The contents of type I collagen and glycosaminoglycan in the epiphysis were increased by administering shark skin gelatin. CONCLUSIONS: These results indicated that shark skin gelatin would be useful as a dietary supplement for treating osteoporosis.

Skeletal developmental effects of selective and nonselective cyclooxygenase-2 inhibitors administered through organogenesis and fetogenesis in Wistar CRL:(WI)WUBR rats.

Cyclooxygenase (COX) inhibitors are the most commonly ingested drugs. The aim of the study was to evaluate the prenatal skeletal effect of selective (DFU) and nonselective (tolmetin, ibuprofen, piroxicam) COX-2 inhibitors. All the tested compounds were administered intragastrically to pregnant Wistar rats from 7 to 21 gestation day. The initial dose was set at 8.5mg/kg/dose for tolmetin and ibuprofen, 0.3 and 0.2mg/kg/dose for piroxicam and DFU. The middle dose was increased 10-times. The highest dose, except for ibuprofen, was elevated 100-times. The highest dose for ibuprofen was set at 200mg/kg/dose. Tolmetin and ibuprofen were administered three times a day. Piroxicam and DFU were dosed once daily. After routine teratological examinations, extremities of randomly selected 21-day-old fetuses were taken for histological, immunohistochemical and molecular studies. The proximal femoral epiphyses were separated and their ultrastructure evaluated. The expression of genes coding cytokines (IL-1alpha, IL-1beta, IL-6, TNF-alpha, TNF-beta) and proteins (COX-1, COX-2, cathepsin K, collagen types I, II and X; osteocalcin, osteopontin) was evaluated in femoral epiphyses by RNase Protection Assay and/or immunohistochemically. The articulate development was checked histologically and found undisturbed in any of the experimental groups. The epiphysis of the 21-day-old fetuses, presented physiological expression of COX-1 and COX-2, as well as cathepsin K, collagen types I, II and X; osteopontin, osteocalcin and TNF-alpha. Increased developmental skeletal variation was noted in groups exposed to the highest dose of nonselective drugs. Unlike the increased number of skeletal variations observed in fetuses exposed to highest doses of nonselective compounds, both groups of COX inhibitors did not disturb joint formation and morphology of femoral epiphyses when administered even in high maternal toxic doses.

[Effects of Qiang Gu Kang Wei prescription on morphology of bone in rats under simulated weightlessness].

OBJECTIVE: To investigate effects of Qiang Gu Kang Wei prescription on bone morphology in tail-suspended rats. METHOD: Rats were tail-suspended (-30 degrees) for 21 d to simulate weightlessness. The effects of this prescription on thickness of periost and cortex at epiphyseal plate of tibial shaft and 3 mm under it were examined. The effects of this prescription on histology and ultrastructure of tibia were also observed. RESULT: Compared with the normal control, cortex at both epiphyseal plate of tibial shaft and 3 mm under it in suspended rats thinned significantly (P<0.01 or P<0.05). Histological observation showed that in bone of suspended rats, uncalcific matrix was more, bone cells' number was lower and their appearance was more infantile, osteoblasts were less and smaller, while osteoclasts were more and larger, haversian system of transection also showed more irregular than in bone of normal control. Ultrastructure observation showed that the function of osteoblasts was inhabited and that of osteoclasts was active. All the three doses groups of the prescription could increase cortex at either epiphyseal plate of tibial shaft or 3 mm under it (P<0.01 or P<0.05) compared with the tail-suspended group. The appearance of bone in the three groups was improved as well under optical or electron microscope. CONCLUSION: This prescription could improve abnormal change in bone morphology resulted from being tail-suspended in rats and make it normal.

Structural skeletal impairment induced by immunosuppressive therapy in rats: cyclosporine A vs tacrolimus.

The exact role of immunosuppressive drugs in the development of osteoporosis and pathologic fractures frequently reported in patients following organ transplantation is still not known. In two experiments, the effects of immunosuppressive drugs were studied on growing rats allocated randomly into five groups of eight rats each which received either FK506 (1.5 mg/kg or 3 mg/kg) or cyclosporine A (15 mg/kg or 30 mg/kg) for 28 days by daily oral gavage. In experiment I ( n=40), bone mineral content (BMC) of the femur by dual energy X-ray absorptiometry (DXA), and bone ash weight were measured. In experiment II ( n=40), stereologic measurements of decalcified tibiae were carried out. The BMC and ash weight values of the whole femur were significantly lower both in the low- and high-dose FK506 groups as well as in the high-dose CsA group. Decalcified sections showed lower volume density of trabecular bone of the tibial metaphysis in both CsA-treated groups and in the high-dose FK506 group. Furthermore, the volume density of the hypertrophic zone volume of the growth plate was higher in high-dose CsA-treated rats. Our data demonstrate that both CsA and FK506 have adverse effects on bone and that high doses of CsA or FK506 alter both cortical and trabecular bone with subsequent osteopenia. In addition, CsA-treated groups showed histological changes in some aspect resembling rickets/osteomalacia.

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.

Safety issues in children and adolescents during growth hormone therapy--a review.

The action of growth hormone (GH) via its receptor involves many organ systems and metabolic pathways. These diverse actions are reviewed in this paper in the context that they may represent unwanted side-effects of GH therapy for growth promotion. The monitoring of GH therapy in large multicentre international databases has demonstrated a low frequency of adverse events. Tumour recurrence or new malignancy are not increased. Headaches, especially in the first few months of therapy, require close evaluation as benign intracranial hypertension is found infrequently, especially in children with GH deficiency and chronic renal failure (CRF). Children at risk for slipped capital femoral epiphysis and scoliosis require close monitoring during therapy. Decreased insulin sensitivity that is dose-dependent is observed during GH therapy. Glucose homeostasis, however, is not affected, but a recent report of increased incidence of Type 2 diabetes mellitus in children undergoing GH therapy requires prospective surveillance.

Generalized metaphyseal modification with cone-shaped epiphyses following long-term administration of 13-cis-retinoic acid.

UNLABELLED: We report on a 6-year-old girl with short stature which developed following the administration of 13-cis-retinoic acid (a synthetic derivative of vitamin A or retinoid) for 40 months as adjunct chemotherapy for neuroblastoma. Radiographic examination suggested osteophyte formation in the cervical spine, which is the most common skeletal manifestation of retinoid toxicity [10, 11]. In addition, severe metaphyseal cupping with a cone-shaped epiphysis primarily affecting rapidly growing long bones was found, which represented impaired enchondral ossification. This epi-metaphyseal alteration, though unusually severe, was reminiscent of the premature epiphyseal closure which has been described as an adverse effect of 13-cis-retinoic acid [10-12]. Other minor skeletal changes included posterior scalloping of the vertebral bodies and increased interpediculate distances, which were related to a widened spinal canal found on CT. A literature search disclosed several primary skeletal dysplasias with superficial radiological similarities to those of the present patient. However, these entities showed significant clinical and radiological differences from our patient. CONCLUSION: The precise cause of the generalized skeletal alteration in the present patient remained unknown, but it conceivably resulted from the administration of 13-cis-retinoic acid.

[Osteogenesis during the correction of burns with alpha-tocopherol]. / Osteogenez pri korrektsii ozhogovoi bolezni al'fa-tokoferolom.

Macro- and microscopic transformations were studied in burned animals skeleton after treatment with alpha-tocopherolum. The antioxidant was found to optimize osteogenesis, disturbed in burn lesion.

Rat epiphyseal cells in culture: responsiveness to bone-seeking hormones.

Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJb medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E2 (PGE2) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE2 (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)2D3 increased significantly the [3H]thymidine incorporation into DNA, whereas 1,25(OH)2D3 effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.

Comparative histological study of the effects of high calcium diet and vitamin D supplements on epiphyseal plates of vitamin-D-deficient chicks.

A comparative histological and microradiographic study of the tibial epiphyseal plates of chickens raised on: (1) a vitamin-D-deficient diet; (2) a vitamin-D-deficient diet supplemented with cholecalciferol, and (3) a vitamin-D-deficient diet to which extra calcium had been added, has revealed that a high-calcium diet did not normalize the epiphyseal plates completely. However, it restored the normal length and chondrocyte arrangement to the proliferative zone. The degenerative zone became elongated and this seems to be related to the hypophosphataemic condition which has developed as a result of the special diet.

Relation between bone mineralization, Ca absorption, and plasma Ca in phosphonate-treated rats.

Disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) is known to inhibit the crystallization of calcium phosphate salts in vitro. Large doses of EHDP administered in vivo inhibit skeletal mineralization, decrease intestinal calcium absorption, and produce hypercalcemia. In the present study, EHDP or one of 13 other phosphonates were given to rats at 10 mg P/kg-day sc for 7 days in order to better define the nature of the relationship between bone mineralization, intestinal absorption, and plasma calcium in the regulation of calcium homeostasis. Each of the phosphonates which inhibited skeletal mineralization in vivo also inhibited crystallization in vitro, but the converse was not true. A very close correlation was found between inhibition of skeletal mineralization, decreased intestinal calcium absorption, and slight hypercalcemia. A dose-response study with two compounds also revealed the same close correlation. It is argued that the impairment of intestinal calcium absorption in phosphonate-treated rats may represent a secondary homeostatic response to the primary effect of the drugs on bone mineralization. This response may be mediated by an elevation of a fraction of plasma calcium.

Action of 1,25-dihydroxyvitamin D3 and a diphosphonate on calcium metabolism in rats.

The effect of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) on Ca balance, 45Ca kinetics, and bone morphology has been studied in control rats and rats given disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), 10 mg P/kg sc per day. This large dose of EHDP is known to inhibit bone mineralization and intestinal calcium absorption and to depress the endogenous production of 1,25-(OH)2D3. In conctrol rats, 1,25-(OH)2D3 increased intestinal calcium absorption. However, in contrast to the enhanced calcium absorption that results from an augmentation of dietary calcium, the 1,25(OH)2D3-induced augmentation of calcium absorption does not lead to a rise in calcium retention, the intestinal effect being matched by an increased excretion of urinary calcium. The EHDP-induced decrease of intestinal calcium absorption could be completely prevented by the concomitant administration of 1,25-(OH)2D3 but not the inhibition of bone mineralization. Therefore, in contrast to the impairment of calcium absorption, that of bone mineralization brought about by large doses of EHDP cannot be merely attributed to a decreased production of 1,25-(OH)2D3.