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In order to generate a calcium-phosphate bone cement as a transient replacement for bone defects, we modified Biocement D (Merck Biomaterial GmbH) containing mineralised collagen with osteocalcin, the most abundant non-collageneous protein of bone. Osteocalcin was added to the cement paste during setting in order to control the crystallisation kinetics of hydroxyapatite (HAP) as well as to stimulate the interaction of osteoblasts and osteoclasts with the bone replacement material. Analysis by SEM and AFM shows, that the addition of osteocalcin causes a nanosize microstructure of the calcium cement, which can be explained by inhibited growth of HAP crystals. The fracture strength of the material decreased by incorporation of osteocalcin, pointing onto a higher defect concentration of the crystalline structure. The impact of osteocalcin onto the interaction of bone cells with HAP-Collagen I-cements was studied in a cell culture system using the human osteosarcoma cell line SAOS-2. Results suggest, that osteocalcin might possibly improve the initial adherence of osteoblast-like cells, whereas proliferation of the cells is not effected.
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PMID:Influence of osteocalcin and collagen I on the mechanical and biological properties of Biocement D. 1220 87

Anorexia nervosa (AN) is increasingly common in adolescent girls and occurs at a time of peak bone mass formation. Osteopenia is common in adolescent girls with AN, and in a cross-sectional study, we have reported low bone formation markers in such girls. To determine the impact of chronic undernutrition on bone mineral accrual in contrast to healthy controls, we prospectively measured bone mineral density (BMD) and body composition by dual energy x-ray absorptiometry, bone metabolism markers, and nutritional and hormonal status at baseline, 6 months, and 12 months in 19 adolescent girls with AN (mean +/- SEM, 15.4 +/- 0.4 yr) and 19 controls of comparable chronological and skeletal age. Overall, nutritional status in subjects with AN improved (mean percentage increase in body mass index from baseline, 9.2 +/- 1.9% and 15.2 +/- 2.6% at 6 and 12 months, respectively), with 11 subjects having recovered weight at 12 months. However, lumbar BMD at 12 months (AN, 0.88 +/- 0.02 g/cm(2), vs. control, 0.98 +/- 0.03 g/cm(2); P = 0.008) remained significantly reduced in AN compared with controls, even in recovered subjects. This was due to significant increases in lumbar BMD in controls vs. no change in AN subjects over the year (0.003 +/- 0.001 g/cm(2).month vs. 0.000 +/- 0.001 g/cm(2).month, respectively; P = 0.04). The most significant determinant of change in lumbar BMD at 12 months was change in lean body mass in both AN (r = 0.62; P = 0.008) and control (r = 0.80; P = 0.0006) groups. There were significant increases in surrogate markers of bone turnover in subjects with AN compared with controls as assessed by osteocalcin (AN, 0.9 +/- 0.4 micro g/liter.month, vs. control, -1.1 +/- 0.4 micro g/liter.month; P = 0.0007), bone-specific alkaline phosphatase (AN, 0.6 +/- 0.5 U/liter.month, vs. control, -1.5 +/- 0.4 U/liter.month; P = 0.002), deoxypyridinoline [AN, 0.1 +/- 0.1 nmol/mmol creatinine (cr).month, vs. control, -0.4 +/- 0.1 nmol/mmol cr.month; P = 0.005], and N-telopeptide (AN, 4 +/- 4 nmol BCE/mmol cr/month, vs. control, -9 +/- 4 nmol BCE/mmol cr/month; P = 0.01). Changes in IGF-I levels over the year were highly correlated with changes in bone turnover over the same period in AN (osteocalcin, r = 0.77; P = 0.001; deoxypyridinoline, r = 0.66; P = 0.01). A rise in N-telopeptide over the year was correlated with an increase in all bone mineral measures, including lumbar bone mineral content (r = 0.58; P = 0.03) and BMD (r = 0.53; P = 0.05) and total bone mineral content (r = 0.69; P = 0.006) and BMD (r = 0.69; P = 0.006) in the AN group. Therefore, despite recovery over 1 yr, poor bone mineral accrual persists in adolescent girls with AN in contrast to rapid bone accrual in healthy girls. Normalization of bone turnover markers occurs in association with nutritional recovery and an increase in the nutritionally dependent bone trophic factor IGF-I. A rise in bone turnover markers may be an early indicator of increase in BMD in recovering girls with AN.
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PMID:Abnormal bone mineral accrual in adolescent girls with anorexia nervosa. 1221 68

Granulocyte colony-stimulating factor (G-CSF) has been shown to affect the biochemical markers of bone metabolism, including serum bone alkaline phosphatase (BALP), serum osteocalcin, and urine deoxypyridinoline. To determine the association between bone resorption and formation and the G-CSF-induced mobilization of peripheral blood stem cells (PBSC), we examined these markers during mobilization in 19 healthy donors. The average (+/- SEM) serum BALP level before treatment was 81.6 +/- 17.0 IU/dL, and the level increased significantly to 117.7 +/- 15.8 IU/dL on day 5 of G-CSF administration (P < .0001). The urine deoxypyridinoline level before treatment was 12.3 +/- 2.4 nmol/mmol creatinine, and this level also increased significantly to 19.4 +/- 3.0 nmol/mmol creatinine on day 5 of G-CSF administration (P < .0001). In contrast, the average level of serum osteocalcin significantly decreased from 8.07 +/- 2.88 ng/mL to 1.53 +/- 0.18 ng/mL on day 5 (P = .0353). During G-CSF administration, we also studied the serum levels of various cytokines (IL-1beta, osteoclastogenesis inhibitory factor [OCIF], IL-6, tumor necrosis factor alpha, transforming growth factor beta, interferon-gamma, macrophage colony-stimulating factor) related to bone metabolism. Only the kinetics of OCIF were significantly affected. The serum level of OCIF increased immediately after the start of G-CSF administration and remained high during G-CSF administration. These results demonstrate that high-dose G-CSF affects bone metabolism and that OCIF may play a role in bone metabolism. Consistent with the notion that G-CSF affects bone metabolism, a significant correlation was observed between CD34+ cell yield and the increase in urine deoxypyridinoline but not for the changes in serum BALP and osteocalcin levels. This result suggests that bone resorption is either directly or indirectly related to the mobilization of PBSC by G-CSF.
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PMID:Effect of granulocyte colony-stimulating factor on bone metabolism during peripheral blood stem cell mobilization. 1256 3

In a recent in vitro study, chemical microroughening of a bioactive glass surface was shown to enhance attachment of MG-63 osteoblastic cells to glass. The current study was designed to delineate the effects of microroughening on the gene expression patterns of bone markers during osteogenesis and new bone remodeling on bioactive glass surface in vivo. With the use of a rat model of paired comparison, a portion of the medullary canal in the proximal tibia was evacuated through cortical windows and filled with microroughened or smooth bioactive glass microspheres. The primary bone-healing response and subsequent remodeling were analyzed at 1, 2, and 8 weeks, respectively, by radiography, pQCT, histomorphometry, BEI-SEM, and molecular biologic analyses. The expression of various genes for bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis of the respective mRNAs. Paired comparison showed significant differences in the mRNAs levels for specific bone matrix components at 2 weeks: osteopontin was significantly higher (p =.01) and osteonectin significantly lower (p =.05) in bones filled with microroughened microspheres than in those filled with smooth microspheres. Bones filled with microrough microspheres also showed significantly increased ratios of cathepsin K and MMP-9 (both markers of osteoclastic resorption) to type I collagen (p =.02 and p =.02, respectively) at 2 weeks and a significantly increased expression of MMP-9 at 8 weeks (p =.05). The pQCT, histomorphometric, and BEI-SEM analyses revealed no significant differences in the pattern of bone-healing response. Based on these results, microroughening of a bioactive glass surface could trigger temporal changes in the expression of specific genes especially by promoting the resorption part of new bone-remodeling processes. Future studies are needed to evaluate if the observed changes of gene expression are directly related to the microrough surface of any biomaterial or are biomaterial specific.
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PMID:Molecular biologic comparison of new bone formation and resorption on microrough and smooth bioactive glass microspheres. 1263 86

The relationship between duration of exercise and serum remodeling markers of bone turnover was evaluated by osteocalcin (OC), carboxy-terminal propeptide of type I collagen (PICP), total and bone-specific alkaline phosphatase (ALP) and carboxyterminal cross-linked telopeptide of type I collagen (ICTP) in 24 male premier league soccer players exercising 12 hours/week (range 8-18), 19 third league players exercising 8 hours/week (range 3-18) and 20 sixth league players exercising 6 hours/week (range 2-10). Twenty-seven volunteers served as controls. Forty-six former male soccer players (mean age 38 years, range 19-47), mean 15 years older than the current players, were compared with 41 matched controls. Data is presented as mean +/- SEM. Active male players had 18 +/- 4% higher OC, 37 +/- 9% higher bone ALP and 36 +/- 7% higher ICTP than controls (all P < 0.01). There were no differences in remodeling markers within the three groups of active players but each group had higher OC and ICTP than controls (both P < 0.05). Former players had no difference in bone remodeling markers compared to matched controls, but 39 +/- 4% lower OC and 69 +/- 8% lower ICTP than active players (both P < 0.001). Duration of activity was correlated with bone ALP and ICTP (both r = 0.3, P < 0.05) in individuals exercising 6 hours/week or less. No correlation was found in those exercising above this level. It seems as if the bone turnover, evaluated by serum bone remodeling markers, adapts to the current activity needed to maintain bone strength, and a duration of exercise above that level seems to confer no additional benefits.
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PMID:The duration of exercise as a regulator of bone turnover. 1287 8

Bone formation was investigated in vitro by culturing rat marrow stromal osteoblasts in biodegradable, macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices over a period of 60 days. Foams were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma (100 W 10 min) to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Osteoblasts were isolated from rat bone marrow and seeded onto PHBV foams. The cell density on and in the foams was determined with MTS assay. MTS results showed that osteoblasts proliferated on PHBV. Twenty-one days after seeding of incubation, growth of osteoblasts on matrices and initiation of mineralization were observed by confocal laser scanning microscopy. Increasing ALP and osteocalcin secretion during 60 days confirmed the osteoblastic phenotype of the derived stromal cells. SEM, histological evaluations and confocal laser scanning microscopy showed that osteoblasts could grow inside the matrices and lead to mineralization. Cells exhibited spindle-like morphology and had a diameter of 10-30 microm. Based on these, it could confidently be stated that PHBV seems to be a promising polymeric matrix material for bone tissue engineering.
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PMID:Bone generation on PHBV matrices: an in vitro study. 1455 13

The aim of the study was to compare bone turnover in male soccer players with controls and to follow bone turnover with changes in activity level. Serum-osteocalcin (OC), carboxy-terminal propeptide of type I collagen (PICP) and total alkaline phosphatases (tALP) were measured to assess bone formation. Bone resorption was detected by carboxyterminal cross-linked telopeptide of type I collagen (ICTP). Bone turnover of 12 male premier league soccer players (mean age 23 years, range, 17-34) exercising 12 hours/week (range, 8-15) were at the last day of the soccer season compared with 27 age- and gender-matched controls. Bone turnover was followed weekly during a 4-week resting period between two seasons, and a further 10 days following resumption of full training. Data are presented as mean +/- SEM. Both OC (22 +/- 12%) and ICTP (34 +/- 17%) were higher in the players compared with the controls at the end of the season (both P < 0.05, respectively). After 2 weeks of reduced physical activity among the athletes, the PICP levels were 21 +/- 4% (P < 0.05) lower and the ICTP levels 8 +/- 12% higher (P = 0.07) compared with baseline. OC, PICP, and tALP was then no different compared with controls and ICTP was higher than controls (P < 0.001). Ten days within the new season, there was a 23 +/- 5% increase in PICP (P < 0.001) and a 4 +/- 4% decrease in ICTP (P < 0.05) compared with the end of the resting period. In summary, male soccer players have higher bone turnover compared with age- and gender-matched controls. Changes in physical activity level were associated with changes in bone formation and resorption as evaluated by bone markers within weeks, and after 2 weeks rest, ICTP was higher in the athletes than the controls. We conclude that the higher age-related diminution in BMD, previously reported in former soccer players compared with age- and gender-matched controls, may be the result of increased bone resorption, evaluated by ICTP, compared with the controls.
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PMID:Bone turnover responses to changed physical activity. 1456 95

In the treatment of osteoporosis, the tolerance of oral bisphosphonates is often low. The high potency of ibandronate allows iv bolus injections that can be repeated every 2 to 3 months. However, the best dose and time interval of the treatment with iv ibandronate is still debated. Efficacy of 2-mg ibandronate injected every 3 months was tested in men with osteoporosis over 2 yr, in a prospective, open study. Fourteen men with primary osteoporosis, mean age 57 +/- 12 yr (range: 40-73), received 2-mg ibandronate iv every 3 months over 2 yr. All got 1 g/day calcium and 880 UI/day vitamin D for 2 yr. Bone mineral density (BMD) increased after 2 yr by 6.7 +/- 1.5% (mean change +/- SEM) at lumbar spine (p<0.001), by 3.2 +/- 08% at trochanter (p<0.001) and by 1.4 +/- 1.1% at femoral neck (ns). Serum beta-crosslaps and osteocalcin decreased significantly by 30-45 and 30%, respectively, during the 2 yr of treatment. Serum calcium increased from the lower to the middle tertile of the normal range during the 2 yr of the study. The observed decrease of bone remodelling and the increase of BMD are of the same magnitude as those described with oral bisphosphonates. The increase of plasma calcium confirms the positive effect of the supplementation with calcium and vitamin D. These results suggest that 3 months are a good interval between two doses of iv ibandronate, when 2 mg are given.
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PMID:Intravenous ibandronate in men with osteoporosis: an open pilot study over 2 years. 1466 26

Electrostatic spray deposition (ESD) is a recently developed technique to deposit a calcium phosphate (CaP) coating upon substrates. With this technique, an organic solvent containing calcium and phosphate is pumped through a nozzle. Between the nozzle and substrate a high voltage is applied. As a consequence, droplets coming out the nozzle disperse into a spray, and this spray is deposited upon the substrate. When the solvent has evaporated, a coating is formed on the substrate. ESD allows for a variation in coating composition and morphology. Titanium alloy (TiAl6V4) substrates were coated with a CaP layer using two different methods; radio frequency magnetron sputtering, and ESD. These surfaces were characterized with X-ray diffraction, Fourier transform infrared spectroscopy, an universal surface tester, scanning electron microscopy, and energy dispersive spectrometry. Subsequently, bone marrow cells were isolated from rat femora and cultured 1, 4, 8, 14 and 16 days. Cell proliferation, alkaline phosphatase activity, and osteocalcin concentration were assayed. RT-PCR was done for collagen type I and osteocalcin. SEM was also performed to observe cellular behaviour during culture. Two separate runs of the experiment were performed. In the first run, osteoblast-like cells on both CaP coatings showed similar results in all assays. In the second run, proliferation and osteogenic expression had increased on ESD coatings. On basis of these results, we conclude that the novel ESD coating behaved similar to, or even better than the known RF magnetron sputter coating. Thus, ESD could be a valid addition to already existing CaP coating processes.
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PMID:Electrostatic spray deposition (ESD) of calcium phosphate coatings, an in vitro study with osteoblast-like cells. 1474 16

Alkaline-heat-treated titanium self-forms an apatite surface layer in vivo. The aim of the present study was to materialistically characterize the surface of alkaline-heat-treated titanium immersed in simulated body fluid (AHS-TI) and to examine the differentiation behavior of osteoblasts on AHS-TI. SEM, thin-film XRD, FTIR, and XPS analyses revealed that AHS-TI contained a 1.0- micro m-thick, low-crystalline, and [002] direction-oriented carbonate apatite surface. Human osteoblast-like SaOS-2 cells were cultured on polystyrene, titanium, and AHS-TI, and RT-PCR analyses of osteogenic differentiation-related mRNAs were conducted. On AHS-TI, the expression of bone sialoprotein mRNA was up-regulated as compared with that on polystyrene and titanium (p < 0.05). On AHS-TI, the expression of osteopontin and osteocalcin mRNAs was up-regulated as compared with that on polystyrene (p<0.05). The results indicate that the apatite was bone-like and accelerated the osteogenic differentiation of SaOS-2, suggesting that alkaline-heat treatment might facilitate better integration of titanium implants with bone.
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PMID:Characterization of apatite formed on alkaline-heat-treated Ti. 1515 53


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