Osteocalcin is one of the most abundant noncollagenous proteins found in adult bone. It is a highly conserved gamma-carboxyglutamic acid-containing protein that is believed to be produced exclusively by osteoblasts. In this study, intracellular and extracellular localization of osteocalcin in osteosarcoma was examined with anti-osteocalcin antibody and in situ hybridization using a synthetic oligonucleotide. Immunohistochemically, osteoblastic osteosarcomas were all positive for osteocalcin. The chondroblastic osteosarcomas were positive on the neoplastic chondrocytes. The five fibroblastic osteosarcomas out of seven were positive for osteocalcin immunostaining over the neoplastic spindle cells. Five cases of osteoblastic osteosarcomas out of seven were positive for osteocalcin in situ hybridization. Two cases of chondroblastic osteosarcomas and three cases of fibroblastic osteosarcomas were positive for in situ demonstration of osteocalcin. The malignant tumor giant cells were positive for osteocalcin immunostaining 83%. They were also positive for in situ hybridization. The benign giant cells in five giant cell tumors and five aneurysmal bone cysts were negative for osteocalcin immunostaining. The benign giant cells in three chondroblastoma and three Paget's disease were positive for osteocalcin. In this study, the osteocalcin in situ hybridization and immunostaining has very important meaning for making differential diagnoses of, especially giant cell rich bone forming tumors.
Base Sequence ; Bone Neoplasms/*chemistry ; Human ; Immunohistochemistry ; In Situ Hybridization ; Molecular Sequence Data ; Osteocalcin/*analysis ; Osteosarcoma/*chemistry

OBJECTIVETo establish a more stable method to isolate osteocytes in vitro, and then to find the differences with osteoblast biological characteristics.

METHODSOsteocytes and osteoblasts were isolated from the bone tissue of 3-day-old rats using sequential collagenase digestion. The cells were identified through cell morphology after 24 hours later. Alkaline phosphatase (ALP) kit was used to stain the first generation cells by Kaplow-way, the bone gla protein (BGP) of the cells were stained by immunocytochemitry. Measured ALP and computed its activity.

RESULTSOsteocytes and osteoblasts showed obviously differences in cell morphology. Osteocytes were star-shaped or dendrite-shaped within more dendrites, while osteoblasts were spindle-shaped with short dendrites. Osteocytes were negative for ALP, but osteoblasts were positive; Osteocytes were more positive for BGP, and osteoblasts were less positive. The secretion of ALP in osteocytes was lower than that of osteoblasts.

CONCLUSIONOsteocytes can be isolated and cultured in vitro. These characteristics of osteocytes are apparently difference with those of osteoblasts.

Alkaline Phosphatase ; analysis ; Animals ; Cell Separation ; methods ; Osteoblasts ; chemistry ; cytology ; Osteocalcin ; analysis ; Osteocytes ; chemistry ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the osteoblast-like functional characteristics exhibited by human periodontal ligament cells (hPDLCs) under mechanical force.

METHODSHuman PDLCs cultured in vitro were stretched by mechanical force. Radioimmunoassay (RIA) was used to measure the expression of secreting alkaline phosphotase (ALP) and osteocalcin (OCN). The non-secreting ALP, OCN and osteopontin (OPN) in cells were determined by immunohistochemistry.

RESULTSIt exhibited increasing of ALP secreted into conditional media, and in the 24 hour period there were two peaks which appeared at the 2nd and 4th hour and the 24th hour (P < 0.01). While in the late of the 24 hours, expression of OCN in conditional media increased (P < 0.05).

CONCLUSIONMechanical force induces hPDLCs to differentiate into functional osteoblast-like cells and plays a role in bone remodeling.

Alkaline Phosphatase ; metabolism ; Cells, Cultured ; Humans ; Osteocalcin ; analysis ; Osteoclasts ; physiology ; Osteopontin ; Periodontal Ligament ; cytology ; Sialoglycoproteins ; analysis ; Stress, Mechanical

To assess the impact of hypogonadism on bone mineral density, we performed a cross-sectional study of 70 amenorrheic women, comprising 22 cases of gonadal dysgenesis and 48 cases of isolated hypogonadotropic hypogonadism (IHH). Bone mineral density was measured by DEXA at four sites: the femur neck, Ward's triangle, trochanter, and lumbar spine (L2-4). The results were compared to those of a control group consisting of 60 age-matched, normal-cycling women. Bone mineral densities around age 20 were already significantly lower at all four sites in patients with IHH and gonadal dysgenesis when compared with controls, suggesting that these patients failed to achieve peak bone mass during pubertal development. In patients with IHH, the initial BMD around age 18-20 were significantly lower at all four sites and the decrease in bone density continued rapidly during the early twenties up to age 25, and then it slowed markedly thereafter. Bone biochemical marker, ICTP and osteocalcin were significantly negatively correlated with age and remained increased until age 40, which was reminiscent of menopausal bone loss pattern such as high bone turn-over in the early twenties, followed by slow bone loss in the late twenties. In patients with gonadal dysgenesis, bone biochemical marker, ICTP and osteocalcin were also significantly negative correlated with age and remained increased until age 40, but no significant changes in BMD were noted as a function of age, which may be attributed to the small sample size and slow bone loss. These findings suggest that the initiation of prompt and timely therapeutic intervention as early as possible in the menarchal period and throughout the remainder of life, particularly during the period associated with rapid bone loss.
Adolescence ; Adult ; Bone Density* ; Collagen/analysis ; Female ; Gonadal Dysgenesis/therapy ; Gonadal Dysgenesis/metabolism* ; Human ; Hypogonadism/therapy ; Hypogonadism/metabolism* ; Osteocalcin/blood ; Peptides/analysis ; Puberty

OBJECTIVETo test the bovine cementoblasts (CBs) cementum-forming ability in vivo.

METHODSRoot fragments of newborn bovine freshly extracted mandibular incisor were cultured routinely and 4th-5th passages of CBs were harvested. CBs were then cultured in the medium supplemented with 50 mg/L alpha-ascorbic acid and 10 mmol/l beta-glycerolphosphate to form a thick layer as tissue engineering scaffold for cementum formation. Collagen membrane was used as control scaffold. 2 x 10(6) cells were attached to the CBs-made carrier as well as collagen membrane scaffolds and transplanted subcutaneously into immunodeficient mice. Transplants were harvested at 7th week. Histological sections were stained with HE, alizarin red S and van Kossa methods as well as monoclonal Ab against bovine cementum attachment protein (CAP).

RESULTSCBs-made scaffold supported more cementum-like tissue (CLT) formation than collagen-made scaffold. The CLT formed on CBs scaffold was partly calcified with embedded cells. Uncalcified cementoid-like material could be seen on the surface and was encircled by cubical CB-like cells. The CLT was also positive to CAP and van Kossa staining.

CONCLUSIONSThese results suggest that the bovine CBs can form cementum-like tissue. The cell-made carrier is a better scaffold than collagen membrane.

Alkaline Phosphatase ; analysis ; Animals ; Bone Transplantation ; methods ; Cattle ; Cell Adhesion Molecules ; analysis ; Cells, Cultured ; Dental Cementum ; chemistry ; cytology ; transplantation ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Osteocalcin ; analysis ; Osteonectin ; analysis ; Sialoglycoproteins ; analysis ; Tissue Engineering ; methods ; Transplantation, Heterologous

OBJECTIVETo examine the changes of osteocalcin and insulin-like growth factor I (IGF-I) during bone lengthening, and to clarify the mechanism of bone healing.

METHODSThirty-two shepherd dogs were divided into five groups randomly. Their tibiae were lengthened by Ilvzarov's external fixator at the rate of 1 mm/day. The lengthening area was the experimental side and the opposite side was the control. Samples were obtained on the 2nd, 4th, 6th, 8th, 12th weekend respectively. The samples were defatted, dried, powdered, centrifuged and measured by radioimmunoassay.

RESULTSThe osteocalcin concentration increased at the subsequent periods, but it was significantly lower in the experimental side than that of the control side, P<0.05 and the IGF-I concentration was not significantly lowered.

CONCLUSIONSDifferent noncollagenous bone growth factors may be different at different stage.

Analysis of Variance ; Animals ; Biomarkers ; analysis ; Bone Lengthening ; methods ; Disease Models, Animal ; Dogs ; Female ; Insulin-Like Growth Factor I ; analysis ; metabolism ; Male ; Osteocalcin ; analysis ; metabolism ; Probability ; Radioimmunoassay ; Random Allocation ; Sensitivity and Specificity ; Statistics, Nonparametric ; Tibia ; surgery

OBJECTIVETo observe effects of blood circulation promoting compounds combined with medicinal guides on content of bone glaprotein (BGP), bone morphogenetic protein-2 (BPM-2) and expression of BMP-2 mRNA in rabbits with femoral head necrosis, and explore its mechanism.

METHODSNinety-eight healthy Spragur-Dawley male rabbits were collected and weighted 2.2 to 2.8 kg. Eighty-four rabbits were built femoral head necrosis model by freezing left femoral head in liquid nitrogen, then randomly divided into 6 groups, 14 in each group. The 6 groups included model group,promoting blood circulation to remove meridian obstruction group,promoting blood circulation to remove meridian obstruction combined with achyranthes bidentata group,radix angelicae pubescentis, asarum group, and platycodon grandiflorum group,other 14 rabbits were sham operation group. While drug groups were administrated corresponding Chinese herb after molding,model group and shamp operation group were given saline. Recombinant human granulocyte-colony stimulating factor ( 30 microg x kg(-1) x d(-1))were injected into all rabbits for 7 days. Samples were taken on the second and fourth week,the content of BGP and BMP-2 were detected by enzyme-linked immunosorbent assay (ELSA) and radioimmunoassay (RIA), histopathological changes of left femoral head were observed by Hematoxylin and Eeosin staining (HE), and expression of BMP-2 mRNA were tested by fluorescence in situ hybridization.

RESULTSCompared with sham operation group, the rate of empty lacunae femoral head were obviously increased in model group, and the content of BGP were increased on the second week, and BMP-2 and BMP-2 mRNA were decreased on the fourth week. Compared with model group, the content of BGP, BMP-2 and BMP-2 mRNA were higher both of the second and fourth week in promoting blood circulation to remove meridian obstruction group. The rate of empty la- cunae femoral head were lower in achyranthes bidentata group, BGP, BMP-2 and BMP-2 mRNA were higher on the fourth week. The rate of empty lacunae femoral head were lower in platycodon grandiflorum group, and BGP were decreased on the second and fourth week, BMP-2 were lower on the second week ,while BMP-2 mRNA were decreased on the fourth week; the content of BMP-2 and BMP-2 mRNA were increased in radix angelicae pubescentis group on the second week; while there was no change in asarum group.

CONCLUSIONRadix angelicae pubescentis can increase the content of BGP, BMP-2 and expression of BMP-2 mRNA ,which is an effective mechanism of preventing femoral head necrosis.

Animals ; Blood Circulation ; drug effects ; Bone Morphogenetic Protein 2 ; analysis ; genetics ; Femur Head Necrosis ; drug therapy ; pathology ; physiopathology ; Male ; Meridians ; Osteocalcin ; blood ; Osteogenesis ; drug effects ; Rabbits

AIMTo explore the effect of hyperhomocysteinemia on vascular calcification and the underlying mechanism of it.

METHODSArterial calcification of Sprague-Dawley rats was induced by vitamin D3 plus nicotine. Hyperhomocysteinemia was established by feeding high methionine diet for six weeks and was assessed b y plasma total homocysteine (tHcy) level detected by HPLC method. Calcification was confirmed by von Kossa staining, measurement of calcium content, alkaline phosphatases (ALP) activity and osteocalcin (OC) concentration of vascular tissue. Lipid conjugated dienes formation were determined to reflecting the production of lipid peroxide.

RESULTSThe results showed that there were mass black granules deposited in aortic wall of the calcified rats by von Kossa staining. Calcium content, ALP activity, OC concentration in calcified rats increased by 8.09-fold, 45.57% and 2.81-fold compared with those of the control group (P < 0.01). Calcium content in calcified rats with high methionine diet increased by 34.29% compared with that of the calcified rats, while ALP activity and OC content decreased by 29.13% and 74.69% compared with that of the calcified rats. Lipid conjugated dienes formation in plasma of the rat with high methionine diet and of calcified rats with high methionine diet increased by 1.93 and 2.89-fold compared with those of the control group, respectively (P < 0.01), and in calcified rats with high methionine diet group was increased by 32.90% compared with that of high methionine diet group (P < 0.01).

CONCLUSIONHyperhomocysteinemia could promote vascular calcification, which might be mediated through the production of lipid peroxide.

Alkaline Phosphatase ; metabolism ; Animals ; Calcium ; metabolism ; Endothelium, Vascular ; Hyperhomocysteinemia ; metabolism ; pathology ; Lipid Peroxidation ; Male ; Methionine ; administration & dosage ; Osteocalcin ; analysis ; Rats ; Rats, Sprague-Dawley ; Vascular Calcification ; metabolism ; pathology

OBJECTIVE: We investigated bone mineral density (BMD) and bone metabolism in female bipolar patients who were undergoing long-term treatment with valproate combined with a low-dose atypical antipsychotic. METHODS: Nineteen premenopausal women with bipolar disorder who were treated with valproate combined with atypical antipsycho-tics for at least 2 years were evaluated. The BMD was measured at lumbar spine and femur sites using dual-energy X-ray absorptiometry (DE-XA). The biochemical markers of bone turnover and circulating levels of gonadal hormones were assessed. Subjects with abnormal DEXA scans were compared to those with normal scans. RESULTS: Nine (47%) of nineteen subjects showed osteopenia or osteoporosis. The T-score for subjects with abnormal DEXA scans was -1.988. Decreased BMD was more prominent in the proximal femur than in the lumbar spine. Subjects with abnormal DEXA scans had high phosphorus and low testosterone levels relative to subjects with normal scans (p=0.008 and p=0.028, respectively). There was a significant negative correlation between phosphorus, osteocalcin, and femur neck BMD (p<0.05). However, multivariate analysis did not show a significant association between femur and lumbar BMD and biochemical markers of bone turnover. CONCLUSION: Long-term treatment with valproate combined with low-dose atypical antipsychotics may adversely affect BMD in premenopausal women with bipolar disorder. A prospective, controlled-study with a larger population is warranted, and assessment of BMD and bone metabolism should be taken into consideration in long-term therapy with valproate and atypical antipsychotics.
Absorptiometry, Photon ; Antipsychotic Agents ; Biomarkers ; Bipolar Disorder ; Bone Density ; Bone Diseases, Metabolic ; Female ; Femur ; Femur Neck ; Gonadal Hormones ; Humans ; Multivariate Analysis ; Osteocalcin ; Osteoporosis ; Phosphorus ; Spine ; Testosterone ; Valproic Acid

OBJECTIVE: To investigate the relationship of polymorphisms in osteoprotegerin (OPG)-receptor activator of NF-kB ligand (RANKL) gene to changes in circulating OPG and soluble RANKL (sRANKL) levels and in bone mineral density (BMD) after hormone therapy (HT) in postmenopausal Korean women. METHODS: The OPG T245G, and G1181C polymorphisms and RANKL rs2277438 A/G polymorphism were analyzed by polymerase chain reaction-restriction fragment length polymorphsim (PCR-RFLP) or direct DNA sequencing in 236 postmenopausal Korean women receiving sequential HT for 1 year. Serum OPG, sRANKL, bone alkaline phosphatase, CrossLaps (CTX), osteocalcin, clacitonin, parathyroid hormone, calicum, and phophorus were measured using enzyme-linked immunosorbent assay (ELISA), immunoassay and atomic absorptiometry respectively. BMD at the lumbar spine and proximal femur was determined by dual energy X-ray absorptiometry. RESULTS: The annual percent changes of BMD were not associated with single or combined genotypes of OPG and RANKL gene polymorphisms, and the distributions of these genotypes were not different between HT-responders and HT-nonresponders (women who lose more than 3% of bone mass per year). After HT of 6 months, change in serum sRANKL levels was significantly higher in GG genotype than in other genotype of RANKL gene polymorphism. No differences in the 6 month changes of other bone turnover markers including circulating OPG levels after HT were noted across single OPG genotype and combined genotypes of OPG and RANKL polymorphisms. CONCLUSIONS: The OPG T245G, and G1181C polymorphisms, and RANKL polymorphism did not associate with change in BMD after HT in postmenopausal Korean women, and RANKL polymorphism affects change in circulating sRANKL levels after HT.
Absorptiometry, Photon ; Alkaline Phosphatase ; Bone Density* ; Enzyme-Linked Immunosorbent Assay ; Female ; Femur ; Genotype ; Humans ; Immunoassay ; NF-kappa B* ; Osteocalcin ; Osteoprotegerin* ; Parathyroid Hormone ; Sequence Analysis, DNA ; Spine