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Journal of Bone and Mineral Research :... Aug 1996Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop...
Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cattle; Drug Stability; Epitope Mapping; Humans; Immunoassay; Linear Models; Osteocalcin; Protein Folding; Recombinant Fusion Proteins
PubMed: 8854253
DOI: 10.1002/jbmr.5650110816 -
BMC Geriatrics Aug 2023Given a lack of studies precisely indicating how many steps elderly people should take daily for their antioxidant defence, bone metabolism, and cognitive abilities to...
BACKGROUND
Given a lack of studies precisely indicating how many steps elderly people should take daily for their antioxidant defence, bone metabolism, and cognitive abilities to improve, our study set out to compare the selected antioxidant, prooxidant, bone turnover, and BDNF indicators between elderly women differing in physical activity (PA) measured by the daily number of steps.
METHODS
The PA levels of 62 women aged 72.1 ± 5.4 years were assessed based on their daily number of steps and then were used to allocate the participants to three groups: group I (n = 18; <5,000 steps a day); group II (n = 22; from 5,000 to 9,999 steps a day); and group III (n = 22; ≥10,000 steps a day). Blood samples were collected from the participants in early morning hours and subjected to biochemical analysis for prooxidant-antioxidant balance indicators (SOD, CAT, GPx, GR, GSH, UA, MDA and TOS/TOC), bone metabolism indicators (Ca, 25-OH vitamin D, osteocalcin, CTX-I, and PTH), and BDNF levels.
RESULTS
The groups were not statistically significantly different in the activity of SOD, CAT, GPx, and GR, but their concentrations of GSH (H = 22.10, p < 0.001) and UA (H = 12.20, p = 0.002) proved to be significantly associated with the groups' daily PA. The between-group differences in the concentrations of MDA and TOS/TOC were not significant, with both these indicators tending to take higher values in group I than in groups II and III. Significant differences between the groups were established for the concentrations of 25-OH vitamin D (H = 24.21, p < 0.001), osteocalcin (H = 7.88, p = 0.019), CTX-I (H = 12.91, p = 0.002), and BDNF (H = 14.47, p = 0.001), but not for Ca and PTH.
CONCLUSIONS
Significantly higher concentrations of GSH, slightly lower oxidative stress indicators, significantly higher BDNF levels, and moderately better bone turnover indicators and resorption markers in the group taking more than 5,000 steps a day suggest that this level of PA can promote successful aging. More research is, however, needed to confirm this finding.
Topics: Female; Humans; Antioxidants; Brain-Derived Neurotrophic Factor; Reactive Oxygen Species; Osteocalcin; Oxidative Stress; Vitamin D; Vitamins; Superoxide Dismutase; Exercise
PubMed: 37580674
DOI: 10.1186/s12877-023-04205-5 -
Physiological Reports Feb 2016Aging is associated with a reduction in osteoblast life span and the volume of bone formed by each basic multicellular unit. Each time bone is resorbed, less is... (Randomized Controlled Trial)
Randomized Controlled Trial
Aging is associated with a reduction in osteoblast life span and the volume of bone formed by each basic multicellular unit. Each time bone is resorbed, less is deposited producing microstructural deterioration. Aging is also associated with insulin resistance and hyperglycemia, either of which may cause, or be the result of, a decline in undercarboxylated osteocalcin (ucOC), a protein produced by osteoblasts that increases insulin sensitivity. We examined whether glucose-loading reduces bone remodeling and ucOC in vivo and osteoblast function in vitro, and so compromises bone formation. We administered an oral glucose tolerance test (OGTT) to 18 pre and postmenopausal, nondiabetic women at rest and following exercise and measured serum levels of bone remodeling markers (BRMs) and ucOC. We also assessed whether increasing glucose concentrations with or without insulin reduced survival and activity of cultured human osteoblasts. Glucose-loading at rest and following exercise reduced BRMs in pre and postmenopausal women and reduced ucOC in postmenopausal women. Higher glucose correlated negatively, whereas insulin correlated positively, with baseline BRMs and ucOC. The increase in serum glucose following resting OGTT was associated with the reduction in bone formation markers. D-glucose (>10 mmol L(-1)) increased osteoblast apoptosis, reduced cell activity and osteocalcin expression compared with 5 mmol L(-1). Insulin had a protective effect on these parameters. Collagen expression in vitro was not affected in this time course. In conclusion, glucose exposure reduces BRMs in women and exercise failed to attenuate this suppression effect. The suppressive effect of glucose on BRMs may be due to impaired osteoblast work and longevity. Whether glucose influences material composition and microstructure remains to be determined.
Topics: Adult; Aging; Blood Glucose; Bone Remodeling; Cells, Cultured; Cross-Over Studies; Exercise; Female; Glucose; Glucose Tolerance Test; Humans; Immunoassay; Insulin; Middle Aged; Osteoblasts; Osteocalcin; Postmenopause
PubMed: 26847728
DOI: 10.14814/phy2.12700 -
Genetic analysis of serum osteocalcin and bone mineral in multigenerational Afro-Caribbean families.Osteoporosis International : a Journal... May 2012Osteocalcin is a major component of bone matrix. Concentrations of total, carboxylated, and uncarboxylated osteocalcin, are highly heritable and genetically correlated...
UNLABELLED
Osteocalcin is a major component of bone matrix. Concentrations of total, carboxylated, and uncarboxylated osteocalcin, are highly heritable and genetically correlated with bone mineral content (BMC) within African ancestry families.
INTRODUCTION
Osteocalcin (OC) is a protein constituent of bone matrix and a marker of bone formation. We characterized the heritability of serum OC measures and identified genomic regions potentially involved in the regulation of OC via high-density genome-wide linkage analysis in African ancestry individuals.
METHODS
African ancestry individuals (n = 459) were recruited, without regard to health status, from seven probands (mean family size = 66; 4,373 relative pairs). Residual heritability of serum OC measures was estimated and multipoint quantitative trait linkage analysis was performed using pedigree-based maximum likelihood methods.
RESULTS
Residual heritabilities of total OC, uncarboxylated OC, carboxylated OC and percent uncarboxylated OC were 0.74 ± 0.10, 0.89 ± 0.08, 0.46 ± 0.10 and 0.41 ± 0.09, respectively. All OC measures were genetically correlated with whole body BMC. We obtained strong evidence of bivariate linkage for percent uncarboxylated OC and whole body BMC on chromosome 17 (logarithm of the odds [LOD] = 3.15, 99 cM).
CONCLUSIONS
All forms of OC were highly heritable and genetically correlated with total body BMC in these African ancestry families. The identified linkage region contains several candidate genes for bone and energy metabolism including COL1A1 and TNFRSF11A. Further studies of this genomic region may reveal novel insight into the genetic regulation of OC and bone mineralization.
Topics: Absorptiometry, Photon; Adolescent; Adult; Aged; Aged, 80 and over; Black People; Bone Density; Female; Genetic Linkage; Genome-Wide Association Study; Genotype; Humans; Male; Middle Aged; Osteocalcin; Quantitative Trait Loci; Young Adult
PubMed: 21935688
DOI: 10.1007/s00198-011-1763-2 -
International Journal of Nanomedicine 2019This study was designed to evaluate the in vitro and in vivo biocompatibility and osteointegration of plasma-sprayed hydroxyapatite (HA)-coated polyethylene...
PURPOSE
This study was designed to evaluate the in vitro and in vivo biocompatibility and osteointegration of plasma-sprayed hydroxyapatite (HA)-coated polyethylene terephthalate (PET) ligaments encapsulated with a simvastatin (SV)-chitosan (CS) composite.
METHODS
This study compared the in vitro and in vivo bone responses to three different PET ligaments: SV/CS/PET-HA, CS/PET-HA and PET-HA. A field emission scanning electron microscope was used to characterize the morphology, and the in vitro SV release profile was analyzed. MC3T3 cells were cocultured with SV/CS/PET-HA, CS/PET-HA and PET-HA to test their biocompatibility using CCK-8 tests. Osteogenic differentiation was investigated by the expression of marker genes using qPCR. Osteointegration was performed by implanting the PET ligaments into the proximal tibia bone tunnels of male Sprague-Dawley rats for 3 weeks and 6 weeks. The bone-implant interface was evaluated by micro-computed tomography (micro-CT) and histological analysis.
RESULTS
The characteristic nanoporous structures mainly formed on the surface of the plasma-sprayed HA particles in the SV/CS/PET-HA and CS/PET-HA groups. The SV release test showed that the sustained release of simvastatin lasted for 25 days in the SV/CS/PET-HA group. The in vitro studies demonstrated that the SV/CS/PET-HA ligaments induced osteogenic differentiation in the MC3T3 cells, with higher mRNA expression levels of collagen-1, bone morphogenetic protein-2, osteocalcin and alkaline phosphatase than those in the CS/PET-HA and PET-HA ligament groups. The in vivo tests showed that both micro-CT analysis (bone mineral density and bone volume per total volume) and histological analysis (bone implant contact and interface area) revealed significantly higher peri-implant bone formation and less interface area in the SV/CS/PET-HA group than in the other groups.
CONCLUSION
The SV-CS composite nanoporous structure was associated with the improved biocompatibility and osteogenic differentiation in vitro and enhanced osteointegration process in vivo of plasma-sprayed HA-coated PET ligaments.
Topics: Animals; Bone and Bones; Cell Differentiation; Cell Line; Cell Proliferation; Chitosan; Drug Liberation; Durapatite; Ligaments; Male; Nanopores; Osseointegration; Osteocalcin; Osteogenesis; Polyethylene Terephthalates; RNA, Messenger; Rats, Sprague-Dawley; Simvastatin; X-Ray Microtomography
PubMed: 31308664
DOI: 10.2147/IJN.S210687 -
Annals of Palliative Medicine Oct 2022Postmenopausal women are one of the most vulnerable groups to osteoporosis. Romosozumab is a newly monoclonal drug that inhibits the activity of sclerostin. Since it has... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Postmenopausal women are one of the most vulnerable groups to osteoporosis. Romosozumab is a newly monoclonal drug that inhibits the activity of sclerostin. Since it has been on the market for only 3 years, there is a lack of systematic analysis on postmenopausal women and the efficacy is not clear. In this study, we compared randomized controlled trials to assess the effects of blosozumab versus placebo in perimenopausal and postmenopausal women.
METHODS
This meta-analysis has been registered in the PROSPERO registry (number CRD42020145839). The PubMed, Cochrane Library, ClinicalKey, and Embase databases were searched from inception date to July 01, 2021. We used the keywords "osteoporosis", "decreased bone mass", and "blosozumab" to retrieve studies on the relationship between blosozumab and osteoporosis in each database. The inclusion criteria were: (I) randomized controlled trials (RCTs) comparing the treatment of osteoporosis with blosozumab and a placebo or without treatment, (II) studies on postmenopausal women aged over 50 years, and (III) studies providing bone mineral density data. The quality of all randomized controlled trials included in this study was independently assessed by two researchers according to the Cochrane risk manual and was divided into high, medium and low quality. The main results analyzed were bone mineral density (BMD) and T-score. Our results mainly include BMD and procollagen type I N-terminal propeptide (P1NP), C-terminal telopeptide of type I collagen (CTX), bone-specific alkaline phosphatase (BSAP), and osteocalcin (OC).
RESULTS
Three RCTs with 105 patients were selected from 157 retrieved articles. Due to high heterogeneity [BMD: Tau2=2.79; Chi2=11.70, degrees of freedom (df) =1 (P=0.0006); I2=91%], we could not perform statistical analysis of BMD. The results of BMD were then evaluated systematically. Three RCT studies were included in the evaluation. Compared with that of the placebo, blosozumab increased levels of the BMD biomarker osteocalcin [mean deviation (MD) 12.55; 95% confidence interval (CI), 8.18, 16.91; P<0.00001]. None of the 3 RCTs presented a risk of bias during the meta-analysis.
CONCLUSIONS
The results suggested that blosozumab could be used as a target drug to improve BMD in postmenopausal women. This will provide a reference for the clinical treatment of postmenopausal women with osteoporosis.
Topics: Female; Humans; Middle Aged; Bone Density Conservation Agents; Osteocalcin; Osteoporosis; Osteoporosis, Postmenopausal; Postmenopause; Randomized Controlled Trials as Topic
PubMed: 36367007
DOI: 10.21037/apm-22-998 -
Methods in Enzymology 2013Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and... (Review)
Review
Determination of protein structure on mineral surfaces is necessary to understand biomineralization processes toward better treatment of biomineralization diseases and design of novel protein-synthesized materials. To date, limited atomic-resolution data have hindered experimental structure determination for proteins on mineral surfaces. Molecular simulation represents a complementary approach. In this chapter, we review RosettaSurface, a computational structure prediction-based algorithm designed to broadly sample conformational space to identify low-energy structures. We summarize the computational approaches, the published applications, and the new releases of the code in the Rosetta 3 framework. In addition, we provide a protocol capture to demonstrate the practical steps to employ RosettaSurface. As an example, we provide input files and output data analysis for a previously unstudied mineralization protein, osteocalcin. Finally, we summarize ongoing challenges in energy function optimization and conformational searching and suggest that the fusion between experiment and calculation is the best route forward.
Topics: Adsorption; Algorithms; Crystallization; Durapatite; Humans; Molecular Docking Simulation; Monte Carlo Method; Osteocalcin; Salivary Proteins and Peptides; Software; Surface Properties
PubMed: 24188775
DOI: 10.1016/B978-0-12-416617-2.00016-3 -
American Journal of Veterinary Research Jan 2004To evaluate a human assay for quantification of carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), assess the influence of age on plasma CTX-I... (Comparative Study)
Comparative Study
OBJECTIVE
To evaluate a human assay for quantification of carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), assess the influence of age on plasma CTX-I concentration, investigate the relationship between plasma CTX-I and serum osteocalcin concentrations, and determine whether concentrations of plasma CTX-I or serum osteocalcin fluctuate in circadian manner in horses. HORSES: 75 clinically normal horses.
PROCEDURE
Cross-reactivity between equine serum CTX-I and CTX-I antibodies in an automated electrochemiluminescent sandwich antibody assay (ECLIA) was evaluated via a specificity test (ie, dilution test) and recovery calculation. Serum osteocalcin concentration was measured with an equine-specific osteocalcin radioimmunoassay. To analyze diurnal variations in plasma CTX-I and serum osteocalcin concentrations, blood samples were obtained hourly during a 24-hour period.
RESULTS
Results of the dilution test indicated good correlation (r > 0.99) between expected serum CTX-I concentrations and measured serum CTX-I concentrations. The calculated CTX-I recovery was 97.6% to 109.9%. Plasma CTX-I and serum osteocalcin concentrations were correlated. Plasma CTX-I concentration was inversely correlated with age of the horse. No significant circadian variations in plasma CTX-I and serum osteocalcin concentrations were detected.
CONCLUSIONS AND CLINICAL RELEVANCE
Results suggest that the fully automated CTX-I ECLIA can be used for evaluation of plasma and serum samples from horses and may be a useful tool to monitor bone metabolism changes. Horses in this study did not have notable diurnal fluctuations in serum osteocalcin and plasma CTX-I concentrations.
Topics: Age Factors; Animals; Circadian Rhythm; Collagen; Collagen Type I; Horses; Immunosorbent Techniques; Luminescent Measurements; Osteocalcin; Peptides; Radioimmunoassay
PubMed: 14719711
DOI: 10.2460/ajvr.2004.65.104 -
Journal of Translational Medicine Oct 2022MicroRNA (miRNA) is accepted as a critical regulator of cell differentiation. However, whether microRNA-223 (miR-223) could affect the osteogenic differentiation of...
BACKGROUND
MicroRNA (miRNA) is accepted as a critical regulator of cell differentiation. However, whether microRNA-223 (miR-223) could affect the osteogenic differentiation of periodontal ligament (PDL)-derived cells is still unknown. The aim of this study was to explore the mechanisms underlying the roles of miR-223 in the osteogenesis of PDL-derived cells in periodontitis.
METHODS
Microarray analysis and real-time polymerase chain reaction (RT-PCR) were used to identify difference in miR-223 expression pattern between healthy and inflamed gingival tissue. The target genes of miR-223 were predicted based on Targetscan and selected for enrichment analyses based on Metascape database. The gain-and loss-of-function experiments were performed to discuss roles of miR-223 and growth factor receptor genes in osteogenic differentiation of PDL-derived cells. The target relationship between miR-223 and growth factor receptor genes was confirmed by a dual luciferase assay. Osteogenic differentiation of PDL-derived cells was assessed by Alizarin red staining, RT-PCR and western blot detection of osteogenic markers, including osteocalcin (OCN), osteopontin (OPN) and runt-related transcription factor 2 (Runx2).
RESULTS
MiR-223 was significantly increased in inflamed gingival tissues and down-regulated in PDL-derived cells during osteogenesis. The expression of miR-223 in gingival tissues was positively correlated with the clinical parameters in periodontitis patients. Overexpression of miR-223 markedly inhibited PDL-derived cells osteogenesis, which was evidenced by reduced Alizarin red staining and osteogenic markers expressions. Furthermore, two growth factor receptor genes, including fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor beta receptor 2 (TGFβR2), were revealed to be direct targets of miR-223 and shown to undergo up-regulation in PDL-derived cells during osteogenesis. Moreover, suppression of FGFR2 or TGFβR2 dramatically blocked PDL-derived cells osteogenic differentiation.
CONCLUSIONS
Our study provides novel evidence that miR-223 can be induced by periodontitis and acts as a negative regulator of PDL-derived cells osteogenesis by targeting two growth factor receptors (TGFβR2 and FGFR2).
Topics: Anthraquinones; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Humans; MicroRNAs; Osteocalcin; Osteogenesis; Osteopontin; Periodontal Ligament; Periodontitis; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Transforming Growth Factor beta
PubMed: 36221121
DOI: 10.1186/s12967-022-03676-1 -
American Journal of Physiology. Cell... Nov 2019Exosome secretion is an important paracrine way of endothelial progenitor cells (EPCs) to modulate resident endothelial cells. The osteocalcin (OCN)-expressing EPCs have...
Exosome secretion is an important paracrine way of endothelial progenitor cells (EPCs) to modulate resident endothelial cells. The osteocalcin (OCN)-expressing EPCs have been found to be increased in cardiovascular disease patients and are considered to be involved in the process of coronary atherosclerosis. Since OCN has been proven to prevent endothelial dysfunction, this study aimed to evaluate the effect of exosomes derived from OCN-overexpressed EPCs on endothelial cells. Exosomes derived from EPCs (Exos) and OCN-overexpressed EPCs (OCN-Exos) were isolated and incubated with rat aorta endothelial cells (RAOECs) with or without the inhibition of OCN receptor G protein-coupled receptor family C group 6 member A (GPRC6A). The effects of exosomes on the proliferation activity of endothelial cells were evaluated by CCK-8 assay, and the migration of endothelial cells was detected by wound healing assay. A tube formation assay was used to test the influence of exosomes on the angiogenesis performance of endothelial cells. Here, we presented that OCN was packed into Exos and was able to be transferred to the RAOECs via exosome incorporation, which was increased in OCN-Exos groups. Compared with Exos, OCN-Exos had better efficiency in promoting RAOEC proliferation and migration and tube formation. The promoting effects were impeded after the inhibition of GPRC6A expression in RAOECs. These data suggest that exosomes from OCN-overexpressed EPCs have a beneficial regulating effect on endothelial cells, which involved enhanced OCN-GPRC6A signaling.
Topics: Animals; Cell Movement; Cell Proliferation; Endothelial Progenitor Cells; Exosomes; Gene Expression; Neovascularization, Physiologic; Osteocalcin; Rats
PubMed: 31411920
DOI: 10.1152/ajpcell.00534.2018