Effects of whole-body vibration on acute bone turnover marker responses to resistance exercise in young men

Objective:

We investigated acute bone turnover marker (BTM) responses to high-intensity resistance exercise with and without whole-body vibration (WBV) in young men (n=10).

Methods:

In this randomized crossover study, subjects performed 2 protocols separated by 2-week wash out periods: 1) resistance exercise only (RE) (3 sets 10 repetitions 80% 1RM for 9 exercises); and 2) WBV + RE (side-alternating vibration platform 5 intermittent, 1-minute bouts 20 Hz, 3.38 mm peak-to-peak displacement followed by RE). Fasting morning blood draws were taken before RE or WBV (PRE), immediately post RE (IP), and 30 minutes post RE (30P). WBV + RE also had a blood draw after the WBV exposure (POST WBV). Blood samples were analyzed for lactate, hematocrit, bone-specific alkaline phosphatase (Bone ALP, U/L), C-terminal telopeptide of type I collagen (CTX-I, ng/mL) and tartrate-resistant acid phosphatase 5b (TRAP5b, U/L).

Results:

Lactate, hematocrit, and Bone ALP significantly increased (p<0.05) IP for both protocols. Bone resorption markers did not change during RE only. CTX-I significantly decreased POST WBV. TRAP5b increased POST WBV, then significantly decreased at 30P.

Conclusions:

Generally, BTM changes to RE only were not significant when adjusted for hemoconcentration. The WBV stimulus altered bone resorption marker but not bone formation marker responses.     

Effects of 0.4 T rotating magnetic field exposure on density, strength, calcium and metabolism of rat thigh bones

The current study investigated the effects of 0.4 T rotary non-uniform magnetic field (RMF) exposure on bone density in ovariectomized (OVX) rats. Results showed that many bone indexes are significantly elevated after RMF exposure compared to the control OVX group and confirmed mechanistic evidence that strong magnetic field (MF) exposure could effectively increase bone density and might be used to treat osteoporosis. Synergy of daily RMF exposure (30 min a day for 30 days using an 8 Hz rotary 0.4 T MF) with calcium supplement tended to increase the indexes of thigh bone density, energy absorption, maximum load, maximum flexibility, and elastic deformation as compared to those of untreated OVX control group. Results also revealed that the indexes of alkaline phosphatase (ALP), serum phosphate, and serum calcium were higher in rats exposed to RMF with calcium than in the untreated OVX control group. Changes in bone mineral density (BMD) and bone mineral content (BMC) were observed in rats for three months including the first month RMF exposure. Bone density in rats exposed each day for 60 min increased during 1-month exposure and continued to increase during the post-exposure period. Furthermore, bone density and calcium content in rats exposed for 90 min daily decreased initially in the exposure month; however, ratio of increase was well above the control values by the end of the post-exposure period suggesting possible window and delayed effects. The study indicated that RMF exposure to both male and OVX female rats for 120 min a day over 15 day period should effectively promote increase of bone calcium contents (BCC) and bone-specific alkaline phosphatase (BAP) in rats thigh bone as well as a corresponding decrease in deoxypyridinoline crosslinks (DPD).     

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.     

The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2.

The possibility that the non-osteogenic mouse pluripotent cell line, C3H10T1/2 (10T1/2), could be induced to differentiate into osteogenic cells by various hormones and cytokines was examined in vitro. Of a number of agents tested, recombinant human bone morphogenetic protein-2 (rhBMP-2) and retinoic acid induced alkaline phosphatase (ALP) activity in 10T1/2 cells. rhBMP-2 also induced mRNA expression of ALP in the cells. Dexamethasone, 1 alpha, 25-dihydroxyvitamin D3, transforming growth factor-beta 1 and insulin-like growth factor-I did not stimulate ALP activity. Treatment with rhBMP-2 greatly induced cAMP production in response to parathyroid hormone in 10T1/2 cells. No ALP activity was induced in NIH3T3 fibroblasts treated with rhBMP-2 or retinoic acid. These results indicate that 10T1/2 cells have a potential to differentiate into osteogenic cells under the control of BMP-2.     

Running inhibits osteoporosis induced by protein-deficient (PD) food intake

Running at 0.7 km/h for 10 min every day inhibited development of osteoporosis caused by protein deficient (PD) food intake. Urine alkaline phosphatase (ALP), a marker of bone formation osteoporosis, was not elevated in rats fed PD, when the osteoporosis was inhibited by running. Estrogen supplementation increased bone-breaking energy (BBE), but did not increase bone mineral density (BMD), and did not decrease urinary ALP levels.     

Small intestinal alkaline phosphatase activity and changes induced by cysteamine administration in fecal excretions of male Wistar rats

Alkaline phosphatase (ALP) activity in fecal excretions was measured in male Wistar rats. Total daily ALP activity in fecal extracts was 133.1 +/- 21.2 mumoles/min per rat weighing approximately 150 g. We found that 63.7% of the fecal ALP activity was inhibited by 30 mM L-phenylalanine (L-Phe), a specific inhibitor for intestinal ALP. As body weight increased from 150 g to 300 g, total daily ALP activity in fecal extracts decreased rapidly to 33.7 +/- 6.08 mumoles/min/rat. However, the percentage of L-Phe-sensitive ALP to total enzyme activity was less variable (40-65%) in the growing rats. Cysteamine-HCl, an ulcerogenic drug, was injected subcutaneously to adult rats (300 g b. w) at a dosage of 400 mg/kg. b. w. Daily excretion of L-Phe-sensitive ALP in fecal extracts decreased to one-third 2 days after injection. Afterwards, a steep and transient increase in the enzyme activity was detected in fecal extracts between days 4 and 7 after injection. ALP activity in fecal excretions may be a clinical indicator of duodenal mucosal damage.     

Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley-Liss, Inc.     

Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor

The use of an amperometric graphite-Teflon composite tyrosinase biosensor for the rapid monitoring of alkaline phosphatase (ALP), with no need of an incubation step and using phenyl phosphate as the substrate, is reported. Phenol generated by the action of ALP is monitored at the tyrosinase composite electrode through the electrochemical reduction of the o-quinone produced to catechol, which produces a cycle between the tyrosinase substrate and the electroactive product, giving rise to the amplification of the biosensor response and to the sensitive detection of ALP. The current was measured at -0.10 V 5 min after the addition of ALP. As a compromise between high ALP activity and high sensitivity for the detection of phenol, a pH of 8.5 was chosen. The substrate concentration was also optimized. A linear calibration plot was obtained for ALP between 2.0 x 10(-13) and 2.5 x 10(-11), with a detection limit of 6.7 x 10(-14) M. Different types of milk were analyzed with good results, using an extremely simple and rapid procedure.     

Fluorescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations

Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone-related protein receptor (PTH1R) as osteoblast markers in dual-color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP(-)PTH1R(-), ALP(+)PTH1R(-), ALP(-)PTH1R(+), and ALP(+)PTH1R(+) populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities ( approximately 150-8,500 cell/cm(2); n = 3). Our results indicate that colony forming unit-osteoblast (CFU-O)/bone nodule-forming cells are found in all fractions, but the frequency of CFU-O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP(-)PTH1R(-), ALP(-)PTH1R(+), ALP(+)PTH1R(-), and ALP(+)PTH1R(+) cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU-O number in the four fractions, with the largest stimulation in the ALP(-) cell populations. However, there was no significant difference in the number or size distribution of CFU-F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non-autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP(-)PTH1R(-) population was capable of forming nodules at the clonal level, at approximately 3- or 12-times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Viability of maxillary bone harvesting by using different osteotomy techniques. A pilot study

The use of autogenous grafts is still considered in bone regeneration surgeries. However, the bone cell viability of such grafts after being harvested from donor sites remains a matter of debate. The aim of the present study is to evaluate particulated and block bone cell viability, in terms of presence or absence of apoptosis and necrosis, obtained from different maxillary intra-oral harvesting methods: bone scraper, rotary carbide burs and piezoelectric device. Five healthy patients were enrolled in the study. The patients required sinus augmentation by lateral window approach. The bone was harvested by the bone scraper, piezoelectric device and rotary surgical instrument. The samples were processed with the Annexin V/FITC (fluorescein isothiocyanate stain) kit and were analyzed by means of Fluoresence-Activated Cell Sorted (FACS) technique. Within the limitations of this pilot study, the results indicated that autogenous bone chips collected from the three harvesting methods presented a large percentage of apoptotic cells, although large scale production of necrotic cells was not detected. In summary, although rotary surgical instrument and piezoelectric devices are frequently used instruments for oral osteotomy, fresh autogenous bone chips collected from them did not present a viable bone cell source.     

Salivary testosterone and cortisol responses in professional rugby players after four resistance exercise protocols

The acute response of free salivary testosterone (T) and cortisol (C) concentrations to four resistance exercise (RE) protocols in 23 elite men rugby players was investigated. We hypothesized that hormonal responses would differ among individuals after four distinct RE protocols: four sets of 10 repetitions (reps) at 70% of 1 repetition maximum (1RM) with 2 minutes' rest between sets (4 x 10-70%); three sets of five reps at 85% 1RM with 3 minutes' rest (3 x 5-85%); five sets of 15 reps at 55% 1RM with 1 minute's rest (5 x 15-55%); and three sets of five reps at 40% 1RM with 3 minutes' rest (3 x 5-40%). Each athlete completed each of the four RE protocols in a random order on separate days. T and C concentrations were measured before exercise (PRE), immediately after exercise (POST), and 30 minutes post exercise (30 POST). Each protocol consisted of four exercises: bench press, leg press, seated row, and squats. Pooled T data did not change as a result of RE, whereas C declined significantly. Individual athletes differed in their T response to each of the protocols, a difference that was masked when examining the pooled group data. When individual data were retrospectively tabulated according to the protocol in which each athlete showed the highest T response, a significant protocol-dependent T increase for all individuals was revealed. Therefore, RE induced significant individual, protocol-dependent hormonal changes lasting up to 30 minutes after exercise. These individual responses may have important ramifications for modulating adaptation to RE and could explain the variability often observed in studies of hormonal response to RE.     

Effects of letrozole on bone biomarkers and femur fracture in female rats

We aimed to investigate the effects of the aromatase inhibitor letrozole on femur fracture and serum levels of alkaline phosphatase (ALP), calcium and phosphate in female rats. Intact 32 Sprague-Dawley female rats were divided into four groups (n=8): control, letrozole 0.2 , letrozole 1 (treatment of 0.2 and 1 mg/kg for six weeks) and recovery (letrozole-treated 1 mg/kg for six weeks then allowed to recover for two weeks). Besides, 24 ovariectomized rats were divided into three groups (n=8): ovariectomized+control, ovariectomized+letrozole and ovariectomized+letrozole+ estradiol (10 μg/rat). After experimental period, rats’ femur bones were removed for biomechanical studies following decapitation. Serum ALP, calcium and phosphate were measured. Biomechanical values, ALP and phosphate significantly increased by letrozole in a dose-dependent manner (p<0.05) while calcium levels and net bone area decreased (p<0.05). Ultimate strength was positively correlated with ALP and phosphate and negatively correlated with calcium. The results indicate that letrozole may increase risk of bone fracture and affect bone biomarkers such as ALP, calcium and phosphate in both intact and ovariectomized rats.     

Strontium ranelate stimulates the activity of bone-specific alkaline phosphatase: interaction with Zn2+ and Mg2+

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl₂) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn²⁺ and Mg²⁺. In the presence of Mg²⁺, both SR and SrCl₂ (0.05–0.5 mM) significantly increased ALP activity (15–66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg²⁺. The cofactor Zn²⁺ also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn²⁺ with 0.05–0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn²⁺ further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.     

Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1–0.4 g at 30 Hz for 10–60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM‐induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM‐induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse‐induced bone loss. J. Cell. Biochem. 106: 306–316, 2009. © 2009 Wiley‐Liss, Inc.     

BMP9经JNK激酶途径调控间充质干细胞C3H10T1/2成骨分化

为了证实JNK激酶在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9) 诱导间充质干细胞C3H10T1/2成骨分化中的作用,利用重组腺病毒将BMP9导入间充质干细胞C3H10T1/2. 通过碱性磷酸酶(ALP)活性测定、钙盐沉积实验、荧光素酶报告基因检测、Western印迹和组织化学染色等方法,检测BMP9是否可经JNK激酶途径调控间充质干细胞C3H10T1/2向成骨分化.动物实验验证在RNA沉默JNK蛋白激酶后,对BMP9诱导间充质干细胞C3H10T1/2向成骨分化的影响．结果发现,BMP9可以增强JNK 激酶的磷酸化;利用JNK抑制剂SP600125抑制JNK激酶活性后,BMP9诱导的间充质干细胞C3H10T1/2的早期成骨指标ALP活性和晚期指标钙盐沉积均受到抑制,而且经典SMAD信号的活化也相应受到抑制;RNA干扰沉默JNK基因表达后,同样也可抑制BMP9 诱导的C3H10T1/2细胞的ALP活性和裸鼠皮下异位成骨．因此表明,BMP9可活化JNK激酶途径从而诱导间充质干细胞C3H10T1/2向成骨分化．