Marker Of Osteoblastic Activity Research Articles

Exposure to Butylparaben Induces Craniofacial Bone Developmental Toxicity in Zebrafish (Danio rerio) Embryos

Butylparaben (BuP) is a common antibacterial preservative utilized extensively in food, medical supplies, cosmetics, and personal care products. The current study reports the use of Zebrafish (Danio rerio) embryos to investigate potential developmental toxicity caused by exposure to BuP. The development of Neural crest cells (NCCs) is highly active during gastrulation in Zebrafish embryos. Thus, we utilized 0.5 mg/L, 0.75 mg/L, and 1 mg/L BuP solutions, respectively, in accordance with the international safety standard dosage. We observed severe craniofacial cartilage deformities, periocular edema, cardiac dysplasia, and delayed otolith development in the Zebrafish larvae 5 days after exposure. The oxidative stress response was significantly enhanced. In addition, the biochemical analysis revealed that the activities of catalase (CAT) and superoxide dismutase (SOD) were significantly reduced relative to the control group, whereas the concentration of malondialdehyde (MDA) was significantly elevated. Furthermore, ALP activity, a marker of osteoblast activity, was also reduced. Moreover, the RT-qPCR results indicated that the expression of chondrocyte marker genes sox9a, sox9b, and col2a1a was down-regulated. In addition, the morphology of maxillofacial chondrocytes was altered in Zebrafish larvae, and the proliferation of cranial NCCs was inhibited. Accordingly, our findings indicate that strong oxidative stress induced by BuP inhibits the proliferation of NCCs in larval Zebrafish, leading to craniofacial deformities.

Rickets guidance: part II—management

Here, we discuss the management of different forms of rickets, including new therapeutic approaches based on recent guidelines. Management includes close monitoring of growth, the degree of leg bowing, bone pain, serum phosphate, calcium, alkaline phosphatase as a surrogate marker of osteoblast activity and thus degree of rickets, parathyroid hormone, 25-hydroxyvitamin D3, and calciuria. An adequate calcium intake and normal 25-hydroxyvitamin D3 levels should be assured in all patients. Children with calcipenic rickets require the supplementation or pharmacological treatment with native or active vitamin D depending on the underlying pathophysiology. Treatment of phosphopenic rickets depends on the underlying pathophysiology. Fibroblast-growth factor 23 (FGF23)-associated hypophosphatemic rickets was historically treated with frequent doses of oral phosphate salts in combination with active vitamin D, whereas tumor-induced osteomalacia (TIO) should primarily undergo tumor resection, if possible. Burosumab, a fully humanized FGF23-antibody, was recently approved for treatment of X-linked hypophosphatemia (XLH) and TIO and shown to be superior for treatment of XLH compared to conventional treatment. Forms of hypophosphatemic rickets independent of FGF23 due to genetic defects of renal tubular phosphate reabsorption are treated with oral phosphate only, since they are associated with excessive 1,25-dihydroxyvitamin D production. Finally, forms of hypophosphatemic rickets caused by Fanconi syndrome, such as nephropathic cystinosis and Dent disease require disease-specific treatment in addition to phosphate supplements and active vitamin D. Adjustment of medication should be done with consideration of treatment-associated side effects, including diarrhea, gastrointestinal discomfort, hypercalciuria, secondary hyperparathyroidism, and development of nephrocalcinosis or nephrolithiasis.

Shatavari Supplementation in Postmenopausal Women Improves Handgrip Strength and Increases Vastus lateralis Myosin Regulatory Light Chain Phosphorylation but Does Not Alter Markers of Bone Turnover.

Shatavari has long been used as an Ayurvedic herb for women’s health, but empirical evidence for its effectiveness has been lacking. Shatavari contains phytoestrogenic compounds that bind to the estradiol receptor. Postmenopausal estradiol deficiency contributes to sarcopenia and osteoporosis. In a randomised double-blind trial, 20 postmenopausal women (68.5 ± 6 years) ingested either placebo (N = 10) or shatavari (N = 10; 1000 mg/d, equivalent to 26,500 mg/d fresh weight shatavari) for 6 weeks. Handgrip and knee extensor strength were measured at baseline and at 6 weeks. Vastus lateralis (VL) biopsy samples were obtained. Data are presented as difference scores (Week 6—baseline, median ± interquartile range). Handgrip (but not knee extensor) strength was improved by shatavari supplementation (shatavari +0.7 ± 1.1 kg, placebo −0.4 ± 1.3 kg; p = 0.04). Myosin regulatory light chain phosphorylation, a known marker of improved myosin contractile function, was increased in VL following shatavari supplementation (immunoblotting; placebo −0.08 ± 0.5 a.u., shatavari +0.3 ± 1 arbitrary units (a.u.); p = 0.03). Shatavari increased the phosphorylation of Aktser473 (Aktser473 (placebo −0.6 ± 0.6 a.u., shatavari +0.2 ± 1.3 a.u.; p = 0.03) in VL. Shatavari supplementation did not alter plasma markers of bone turnover (P1NP, β-CTX) and stimulation of human osteoblasts with pooled sera (N = 8 per condition) from placebo and shatavari supplementation conditions did not alter cytokine or metabolic markers of osteoblast activity. Shatavari may improve muscle function and contractility via myosin conformational change and further investigation of its utility in conserving and enhancing musculoskeletal function, in larger and more diverse cohorts, is warranted.

SAT-356 Reversible Suppression of Serum 1,25-Dihydroxyvitamin D in Williams Syndrome

Background: Hypercalcemia has been reported in 5% to 50% of patients with Williams syndrome; however, observations about the role of 1,25-dihydroxyvitamin D in hypercalcemia related to Williams syndrome have been contradictory. Objective: The objective was to investigate the mineral metabolism in an 11-month-old patient with Williams-Beuren syndrome who had hypercalcemia, hypercalciuria and nephrocalcinosis. Methods: We reviewed test results in an 11-month-old infant with Williams syndrome who developed hypercalcemia following excessive dietary calcium intake that was two to three times higher than recommended for age because he continued receiving a premature formula with high calcium content from birth. Results: Our patient presented with feeding difficulties, daily vomiting, weight loss, and constipation. He had serum total calcium 14.8 mg/dL, phosphorus 4.0 mg/dL, PTH <4 pg/mL, 1,25-dihydroxyvitamin D <8 pg/mL, 25-hydroxyvitamin D 29 ng/mL, PTH-related peptide 3.7 pmoL/L (expected <4.2), alkaline phosphatase 118 U/L, beta-crosslaps 1257 pg/mL, and urinary calcium/creatinine ratio 1.19. XR Skeletal Survey revealed increased bone density in the metaphyseal regions, in particular, above the knee, distal radius and ulna that may result from hypercalcemia; no evidence of rickets, osteoporosis or congenital osteodystrophy was seen. Renal ultrasound revealed increased echogenicity of the renal medullary pyramids consistent with medullary nephrocalcinosis. Hypercalcemia and hypercalciuria were initially treated with IV fluids and Furosemide IV, and resolved only after Pamidronate 0.5 mg/kg/dose IV x 2 doses. After serum calcium normalized, the patient’s symptoms resolved, PTH recovered to 18 pg/mL (expected 10 - 65), and 1,25-dihydroxyvitamin D increased to 27 pg/mL (expected 24 - 86). Chromosomal micro-array analysis showed a 1.9 megabase deletion at 7q11.23 that overlapped the Williams syndrome critical region including the ELN gene, consistent with diagnosis of Williams syndrome. Serum calcium remained normal with dietary calcium restriction using a low-calcium formula and complementary foods. Conclusions: PTH-independent hypercalcemia in our patient with Williams syndrome was due to calcium overload resulting from dietary calcium excess in addition to possibly enhanced intestinal calcium absorption. Serum 1,25-dihydroxyvitamin D was undetectable and returned to normal only with resumption of PTH secretion required for its synthesis after hypercalcemia resolved. The balance between bone formation and resorption was shifted to resorption possibly to remove skeletal calcium excess, based on normal alkaline phosphatase (marker of osteoblast activity) and high collagen beta-crosslaps (marker of osteoclast activity). A premature formula with high calcium content should be switched to a full-term formula when risk for rickets of prematurity clears.

The Effect of Isometric Exercise Plantar Flexor on Osteoblast Activity Measured by Bone Specific Alkaline Phosphatase and Callus Formation in a Patient Post Open Reduction Internal Fixation with Non-articular Tibia Fracture.

BACKGROUND:Post-operative non-articular tibia fracture several problems that can occur include; pain, muscles atrophy, muscles weakness, joint stiffness, delayed union, and non-union that makes patients limited in their daily activities. Several factors that play a role in the process of fracture healing include osteoblast activity and exercise. Isometric exercises plantar flexor might effectively prevent the problem. Markers of osteoblast activity are bone-specific alkaline phosphatase (BSAP) levels and Hummer scale callus formation. Not yet known the effect isometric exercise of plantar flexor on osteoblast activity in the post open reduction internal fixation of non-articular tibia fractures, due to the lack of studies in this field.AIM:This research was conducted to investigate the effect of isometric plantar flexor on osteoblast activity and callus formation in patients post open reduction internal fixation nonarticular tibial fractures.HYPOTHESIS:There are differences in osteoblast activity and callus formation between groups that do isometric exercises of plantar flexor muscles with those that without isometric exercises plantar flexor.ANALYSIS:Hypothesis test used a paired t-test with a value of α 0.05 and a confidence level of 95%.METHODS:This clinical trial was true experimental with pre-post test control group design divided into two groups, group I obtained treatment of isometric exercises of the plantar flexor muscle, range of motion knee joint, and ankle while group II obtained the range of motion knee joints and ankle. Osteoblast activity measured with bone-specific alkaline phosphatase level and callus formation.RESULTS:The result of the study found to increase mean bone-specific alkaline phosphatase group I; 15.6 and group II; 5.2. A paired t-test of independent samples with α of 0.05 and confidence interval 95% was obtained p-value = 0.000, there is a significant difference in increased levels of bone-specific alkaline phosphatase group I obtained isometric exercises plantar flexor with group II without isometric exercises plantar flexor in patients post open reduction internal fixation of a non-articular tibia fracture. Radiographic examination of Hummer scale callus in group I who carried out isometric exercises plantar flexor had an average value of 2.63, whereas group II without isometric exercises plantar flexor average of 3.06. Wilcoxon test with α 0.05 and 95% confidence interval obtained p-value = 0.000, there is a significant difference in callus image in both groups of patients post open reduction internal fixation non-articular tibia fracture. The linear regression calculated of callus assessment with bone-specific alkaline phosphatase obtained the value of R quadrat = 0.793, which showed that the formation of callus Hummer classification could predict the change in bone-specific alkaline phosphatase value by 79%.CONCLUSION:The research found isometric exercise plantar flexor in patients post open reduction internal fixation non-articular tibia fracture enhances osteoblast activity and callus formation that will likely short the healing process time and prevent delayed union or non-union.

Abstract 3671: Phenotypic heterogeneity within prostate cancer bone metastases measured by 18F-DCFBC PET/CT and 18F-NaF PET/CT

Abstract Purpose: The purpose of this study was to compare the spatial colocalization of prostate-specific membrane antigen (PSMA) targeting agent 18F-DCFBC PET/CT, a marker which demonstrates correlation with tumor aggressiveness, and 18F-NaF PET/CT, a marker of osteoblastic activity, within bone lesions of patients with metastatic prostate cancer. Methods: Twenty-eight metastatic prostate cancer patients received 18F-DCFBC PET/CT and 18F-NaF PET/CT scans (median interval 7 days, range 1-57). Tracer-specific regions of interest (ROI) were derived from threshold containing 80% of maximum SUV (SUV80%). Lesion detection and uptake was compared across the two PET/CT agents using non-parametric testing (Spearman correlation, Wilcoxon rank sum) accounting for intra-patient correlation. Lesion-specific registration was completed by global alignment of skeletal segments followed by local neighborhood optimization of area surrounding lesion. Spatial concordance was evaluated using overlap volume (OV), calculated as the volume of ROI intersection (shared volume) relative to the minimum volume of NaF-ROI or PSMA-ROI to account for differences in ROI volumes. Differences in uptake and CT Hounsfield units (HU) were compared within concordant (overlapping) and discordant (NaF-only, PSMA-only) regions of the same lesion using Wilcoxon signed rank test accounting for intra-patient correlation. Results: Twenty-six patients had positive NaF or DCFBC PET/CT scans, with a total of 241 metastatic sites identified. Lesion-level NaF SUV80% and DCFBC-2hr SUV80% were not significantly correlated at baseline (ρ=0.41, p&amp;gt;0.1). Lesion-level NaF-SUV80% was lower in bone lesions detected only by NaF (N=96) compared to NaF-SUV80% in bone lesions detected by both tracers (N=82), nearing significance (p=0.065). This pattern was not observed in DCFBC-SUV80% (p=0.5). To avoid bias from motion artifacts, only lesions in pelvis and spine were considered for voxel-based analysis (N=42). NaF and PSMA ROIs showing varying levels of spatial concordance (OV mean=0.26, range=0-1), which was modestly associated with higher sclerosis (ρ=0.4, p=0.01) and weakly associated with lower PSMA activity (ρ=0.3, p=0.07). Intra-lesion PSMA-only regions were significantly less sclerotic compared to concordant (p=0.04) and NaF-only (p=0.03) regions, indicating regions of active tumor seeding extend beyond regions of active bone remodeling within the tumor. Conclusions: Metastatic seeding and growth of prostate cancer in bone is biologically complex and dependent on bone microenvironment. We find regions of aggressive cellular activity (PSMA-targeting PET/CT) show heterogeneous colocalization with regions of rapid bone remodeling (NaF PET/CT) within sites of metastatic bone disease. These preliminary findings are important for consideration of targeted radionuclide therapies in metastatic prostate cancer. Citation Format: Stephanie A. Harmon, Esther Mena, Joanna Shih, Ethan Bergvall, Stephen Adler, Sherif Mehralivand, Ravi A. Madan, James L. Gulley, William L. Dahut, Baris Turkbey, Peter L. Choyke, M Liza Lindenberg. Phenotypic heterogeneity within prostate cancer bone metastases measured by 18F-DCFBC PET/CT and 18F-NaF PET/CT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3671.

The Relevance of Osteoclastic and Osteoblastic Activity Markers Follow-Up in Patients on Antiresorptive Osteoporosis Treatment

In general, markers of bone formation and markers of bone resorption are changing synergistically, so the monitoring of any osteoclastic and any osteoblastic marker should reflect the rate of bone transformation. The aim of the study is to monitor the bone metabolism markers in postmenopausal women with osteoporosis and osteopenia along with the variations caused by the effects of bisphosphonate therapy. The study involved 55 women of average age of 57.95 years, with osteopenia or osteoporosis. The patients with osteoporosis were treated with bisphosphonates (75 mg once a week); the laboratory tests were performed before the treatment and 6 months later. Patients with osteopenia were evaluated at the first assessment and 6 months later. The tests included bone densitometry, dual-energy X-ray absorptiometry, osteocalcin, alkaline phosphatase, collagen 1 N-terminal pro-peptide (P1NP), and beta C telopeptide of type I collagen (CTX). The mean T-score was −2.80 ± 0.63 before therapy and −2.64 ± 0.45 6 months later (p < 0.001). Women with osteoporosis had elevated levels of osteocalcin and P1NP at the first assessment, whereas the alkaline phosphatase level did not change with the treatment. After the introduction of antiresorptive therapy, the levels of osteocalcin and P1NP significantly decreased (p < 0.001). In the group with osteopenia, the biochemical markers activity were increased in both assessments. In patients with osteoporosis, Beta-CTX was increased in the first evaluation, and decreased after treatment (p = 0.001). The results indicate that the assessment of biochemical markers of bone metabolism show excellent results in the assessment of prognosis, monitoring the course and the response to various treatment regimens of osteoporosis and evince strong correlation with standard densitometry and dual-energy X-ray absorptiometry procedures. P1NP and CTX show better diagnostic applicability compared with osteocalcin and alkaline phosphatase. The analysis of the activity of biochemical markers may obtain early information on the therapeutic response, before definitive assessment by bone density measurements.

Regulation of Osteoblast Function in Myeloma Bone Disease By Semaphorin 4D

Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D.We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease.After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression.Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. DisclosuresRoodman:Amgen: Consultancy.

RANKL promotes osteoblastic activity in vascular smooth muscle cells by upregulating endothelial BMP-2 release

IntroductionReceptor activator of nuclear factor kappa beta-ligand (RANKL) is thought to promote vascular calcification (VC) by inducing osteoblastic behaviour in vascular smooth muscle cells (VSMC) in an ill-defined process. The present study assessed whether RANKL affects pro-osteoblastic paracrine signalling between human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC) using both conditioned media transfer and cell co-culture experimental approaches. Methods and resultsFor initial experiments (6-well format), HAEC-conditioned media was harvested following 72h exposure to RANKL, and transferred to reporter HASMCs with/without noggin, an inhibitor of pro-osteoblastic bone morphogenetic protein (BMP) paracrine signalling. In further experiments, HAECs and HASMCs were co-cultured within the CellMax® Duo, a perfusing bioreactor unit that mimics the flow-mediated co-interaction of these cells within the arterial wall, and RANKL was added to the perfusing media for 72h. At the conclusion of each experiment markers of osteoblastic activity were measured in HASMCs, including alkaline phosphatase (ALP) activity, mRNA levels of ALP and Runx2, as well as BMP-2 and BMP-4 concentrations. RANKL increased BMP-2 release from HAECs, while exposure of HASMCs to RANKL-treated HAEC-conditioned media induced osteoblastic behaviour in HASMCs, an effect prevented by noggin. Within the CellMax® Duo bioreactor, the addition of RANKL to the intraluminal HAECs also produced an increase in BMP-2 and increased osteoblastic behaviour within the co-cultured HASMC population. ConclusionsRANKL promotes VC by inducing BMP-2 release from HAECs, which in turn appears to act in a paracrine fashion on the adjacent HASMC population to increase osteoblastic activity.

The effects of acute hyperinsulinemia on bone metabolism

Insulin signaling in bone-forming osteoblasts stimulates bone formation and promotes the release of osteocalcin (OC) in mice. Only a few studies have assessed the direct effect of insulin on bone metabolism in humans. Here, we studied markers of bone metabolism in response to acute hyperinsulinemia in men and women. Thirty-three subjects from three separate cohorts (n=8, n=12 and n=13) participated in a euglycaemic hyperinsulinemic clamp study. Blood samples were collected before and at the end of infusions to determine the markers of bone formation (PINP, total OC, uncarboxylated form of OC (ucOC)) and resorption (CTX, TRAcP5b). During 4 h insulin infusion (40 mU/m2 per min, low insulin), CTX level decreased by 11% (P<0.05). High insulin infusion rate (72 mU/m2 per min) for 4 h resulted in more pronounced decrease (−32%, P<0.01) whereas shorter insulin exposure (40 mU/m2 per min for 2 h) had no effect (P=0.61). Markers of osteoblast activity remained unchanged during 4 h insulin, but the ratio of uncarboxylated-to-total OC decreased in response to insulin (P<0.05 and P<0.01 for low and high insulin for 4 h respectively). During 2 h low insulin infusion, both total OC and ucOC decreased significantly (P<0.01 for both). In conclusion, insulin decreases bone resorption and circulating levels of total OC and ucOC. Insulin has direct effects on bone metabolism in humans and changes in the circulating levels of bone markers can be seen within a few hours after administration of insulin.

Study of the viability and adhesion of osteoblast cells to bone cements mixed with hydroxyapatite at different concentrations to use in vertebral augmentation techniques

ObjectiveThe purpose of this study is to compare the biocompatibility and the effect in osteoblasts of polymethyl methacrylate alone, and mixed with hydroxyapatite in different concentrations of 5, 10, 15 and 20%, without exceeding 20%, as it can alter mechanical properties of the composite. Materials and methodsExperimental study comparing osteoblast response to polymethyl methacrylate alone and with hydroxyapatite in different concentrations. ResultsComposites at 15 and 20% obtained better osteoblast response, with higher osteoblastic activity markers, and lower apoptosis markers. Electron microscopy images show improved adhesion of osteoblasts.

Estudio de viabilidad y adhesividad celular de osteoblastos a cementos óseos utilizados en técnicas de refuerzo vertebral, mezclados con hidroxiapatita a distintas concentraciones

ObjectiveThe purpose of this study is to compare the biocompatibility and the effect in osteoblasts of polymethyl methacrylate alone, and mixed with hydroxyapatite in different concentrations of 5, 10, 15 and 20%, without exceeding 20%, as it can alter mechanical properties of the composite. Material and methodsExperimental study comparing osteoblast response to Polymethyl methacrylate alone and with hydroxyapatite in different concentrations. ResultsComposites at 15 and 20% obtained better osteoblast response, with higher osteoblastic activity markers, and lower apoptosis markers. Electron microscopy images show improved adhesion of osteoblasts.

Is tartrate-resistant acid phosphatase 5b a potent bio-marker for late stage aseptic implant loosening?

Aseptic loosening is a serious complication after total joint arthroplasty. Plain radiography, along with clinical signs of prosthesis loosening, is the technique of first choice to evaluate loosening of joint replacements. Nevertheless, radiographical signs of osteolysis may not be apparent until progressed stages of loosening. Thus the search for alternative diagnostic methods is of great importance. The purpose of the present study was to evaluate the potential diagnostic significance of TRAP 5b, Osteocalcin, CrossLaps and Bone ALP for aseptic loosening of total joint replacements. Thirty-seven patients (25 women, 12 men, mean age 65 years, age range 54-76 years) treated with revision surgery due to clinically and radiologically confirmed late aseptic prosthesis loosening were examined prospectively. Serum levels of TRAP 5b, Osteocalcin, CrossLaps and Bone ALP were compared with a matched control group (n = 39). We found a significant decrease in TRAP 5B level in patients with aseptic loosening. Bone ALP and Osteocalcin as markers of osteoblast activity, and CrossLaps as another resorption marker did not allow any prediction of bone remodeling in this late phase of loosening. In the "late" phase of aseptic joint replacement loosening, no increase of TRAP 5b and therefore no increase of osteoclast number and activity was measurable. Thus, in this situation an anti-osteolytic therapy with a bisphosphonate or denosumab would not gain any further benefit.

High Bone Mass in Mice Lacking Cx37 Because of Defective Osteoclast Differentiation

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.

Bone Mineral Density and Biochemical Markers of Bone Turnover During the First Year of Injury in Patients with Spinal Cord Injury

BackgroundSpinal cord injury (SCI) is associated with bone mass loss that can be complicated by fractures, which result in further disabilities for patients. After a SCI, the body starts losing large amounts of calcium and other minerals in the urine (demineralisation). This study aimed to assess the changes in bone mineral density (BMD) during the 1st year of acute SCI in patients with neurological deficit. MethodsA total of 95 patients with acute SCI and neurological deficit were evaluated in this prospective study. Haematological investigations such as evaluation of serum calcium, serum phosphate, serum creatinine, and serum alkaline phosphatase (ALP) were carried out. Urinary investigations such as 24-hour urinary creatinine level and excretion of calcium and phosphate in the urine were measured. BMD was measured using dual-energy X-ray absorptiometry scan with Hologic QDR 2000 scanner (Explorer). All of the aforementioned parameters were measured again at 3, 6, and 12 months. ResultsSerum ALP at 1-year follow up was significantly raised (p<0.05). The BMD at 1-year follow up had statistically significant lower values than the initial BMD at the hip (p<0.05), proximal tibia (p < 0.00l), and distal tibial epiphysis (p < 0.001). The BMD in motor-complete SCI patients [American Spinal Injury Association (ASIA) grades A and B] had significant lower values than motor-incomplete SCI patients (ASIA C and D) at the hip (p < 0.05) and proximal tibial epiphysis (p < 0.05). ConclusionThere was a marked decrease in BMD in metaphyseal sites than below the neurological deficit level with maximum decrease at the proximal tibia during the 1st year of SCI. Although the markers of osteoblastic activity did not show much change, the decrease in BMD was influenced by the neurological recovery after SCI.

A Transgenic Model Of Multiple Myeloma Bone Disease Shows Profound Mesenchymal Stem Cell Impairment

Bone disease affects 70% of Multiple Myeloma (MM) patients during the course of their illness. While new treatments for the MM itself have prolonged their survival, patients are living longer with their bone disease. Bisphophonates have been shown to reduce skeletal related events in MM, but they neither eliminate these events nor reverse skeletal damage. The transgenic mouse model of MM (vk*myc) is the only mouse model that has been shown to faithfully recapitulate the clinical disease, including its bony abnormalities. The original publication of the model showed principally a decrease in bone mineral density of affected animals but scant lytic lesions. We have cross bred this model with a substrain of C56/BL6 mice, KaLwRiJ, that is the basis of a competing model of MM and its bone disease the 5T33 model. This new transgenic substrain shows significant number bony lesions on x-ray and microCT analyses. Immunohistochemistry of the femurs and spines of these animals show an increased number of osteoclast and a decreased number of osteoblasts as compared to non-transgenic wild type mice of the same substrain. Dynamic labeling of bone shows a decreased mineral apposition rate (1.21mm/day ±0.03 vs 2.05mm/day ±0.04) in mice with MM vs wild type mice. In addition to the demineralization associated with MM bone disease and unique to this model, we show a dramatic decrease in the formation of osteoblasts from cultured mesenchymal stem cells (MSCs). These are grown ex-vivo from affected transgenic mice and induced to differentiate into osteoblasts in culture. We demonstrate a close to 90% reduction in observed and quantified mineralized colonies indicating a dramatic impairment in MSC differentiation in these mice, similar to what is seen in human MM. Gene expression profiling analyses using mRNA from ex-vivo mesenchymal stem cells derived from transgenic and wild type mice reveal a number of pathways and genes that can potentially play a role in the inhibition of MSC differentiation in this model. These include the wnt signaling pathway as well as genes involved in histone acetyltransferase activity. The latter suggests that MSCs are ‘permanently' affected by the presence of MM cells in vivo and that this inhibition does not improve even when they are separated from MM cells for a prolonged period of time. Bortezomib has been shown in MM patients to improve the appearance of MM lytic lesions radiographically as well as to improve serum markers of osteoblastic activity. This improvement is thought to be due to a reversal of MSC differentiation impairment. Wild type and transgenic mice with MM and bone disease were treated with bortezomib (0.5mg/Kg twice per week) for two weeks. CT analyses of mice pre and post bortezomib treatment showed a 28% improvement in the treated transgenic mice. We also show a concomitant improvement in the ex-vivo ability of MSCs to differentiate into osteoblasts. In summary, we present a unique animal model to study MM bone disease that shows profound MSC impairment. This model responds to a treatment strategy that has been shown to work in the human disease and opens itself to further study and use for future developments targeting MM bone disease. Disclosures:No relevant conflicts of interest to declare.

IGF-I increases markers of osteoblastic activity and reduces bone resorption via osteoprotegerin and RANK-ligand

BackgroundBone is one of the major target tissues for Insulin-like Growth Factor I (IGF-I). Low doses of IGF-I were able to improve liver-associated osteopenia. In the present work, a model of partial IGF-I deficiency was used in order to provide insight into the mechanisms of the beneficial actions of IGF-I replacement therapy in bone.MethodsSeveral proteins involved in osteoblastic/osteocyte and osteoclastic differentiation and activity were studied in the three experimental groups: control (CO) group (wild type mice, Igf+/+, n = 10), heterozygous Igf+/- group with partial IGF-I deficiency (Hz, n = 10), and heterozygous Igf+/- mice treated with IGF-I for 10 days (Hz + IGF-I, n = 10).ResultsData in this paper confirm that the simple partial IGF-I deficiency is responsible for osteopenia, determined by densitometry and histopathology. These findings are associated with a reduced gene expression of osteoprotegerin, sclerostin, calcitonin receptor (CTR), insulin-like growth factor binding protein 5 and RUNX2. IGF-I replacement therapy normalized CTR gene expression and reduced markers of osteoclastic activity.ConclusionsLow doses of IGF-I constituted a real replacement therapy that normalized IGF-I serum levels improving the expression of most of these proteins closely involved in bone-forming, and reducing bone resorption by mechanisms related to osteoprotegerin, RANKL and PTH receptor.

Plasma Rich in Growth Factors Promotes Bone Tissue Regeneration by Stimulating Proliferation, Migration, and Autocrine Secretion in Primary Human Osteoblasts

Alveolar bone loss can be a major clinical concern affecting both functionality and esthetics. Osteoblasts are the main cells charged with the repair and regeneration of missing bone tissue. Plasma rich in growth factors (PRGF) allows delivery of a cocktail of proteins and growth factors that promote wound healing and tissue regeneration to the site of injury. This study tests the effect of this endogenous regenerative technology to stimulate alveolar osteoblast bone-forming potential. Primary human osteoblasts were retrieved from alveolar bone of patients undergoing oral surgery. Cell proliferation was evaluated, and culture inserts and permeable transwell supports were used to assess cell migration and chemotaxis. The expression of differentiation markers was quantified by enzyme-linked immunosorbent assay. PRGF succeeded in increasing proliferation, migration, and chemotaxis of osteoblasts. Also, PRGF significantly enhanced the autocrine expression of two relevant proangiogenic factors, vascular endothelial growth factor and hepatocyte growth factor, and three markers of osteoblastic activity, procollagen I, osteocalcin, and alkaline phosphatase. The results indicate that PRGF can stimulate some of the biologic processes of the main cells responsible for bone regeneration and help support the positive clinical outcomes that have been reported with this technology.

Dietary tryptophan manipulation reveals a central role for serotonin in the anabolic response of appendicular skeleton to physical activity in rats

Several studies support a serotonin role in the physiological control of bone mass. However, whether serotonin (5-HT) is involved in bone loss due to reduced mechanical stress or unloading is unknown. We investigated the effects of reduced 5-HT tone, induced by tryptophan-free diet, in movement-restraint osteopenia induced by housing mature rats, acclimatised in single cages with a floor area of 1,500 cm(2), in smaller size single cages where their motor activity was reduced. Tryptophan-deficiency significantly worsened movement-restraint-induced bone loss in both femoral metaphysis and diaphysis (DXA analysis) but not at lumbar vertebrae and impaired the mechanical properties of the femur by significantly reducing both cortical thickness and strength strain index (pQCT analysis). Such effects resulted from an impairment of bone turnover with bone resorption exceeding bone formation. Tryptophan-supplemented diet reversed the worsening effects of tryptophan-deficiency on movement-restraint osteopenia. The improvements of both bone mass and strength were associated with an increase of serum osteocalcin and IGF-I, markers of osteoblast activity. In vitro studies in primary cultures of rat osteoblasts suggest that the anabolic action of 5-HT involves the activation of the Wnt/β-catenin pathway. Serotonin significantly increased the cytoplasmatic β-catenin protein levels by the inhibition of the enzyme glycogen synthase kinase-3β, that by phosphorylating β-catenin promotes its degradation. Our data support a role for 5-HT in the anabolic response of the appendicular skeleton to mechanical loading. We suggest that serotonin might stimulate canonical Wnt/β-catenin-dependent bone formation to occur.

A cross‐sectional study of osteocalcin and body fat measures among obese adolescents

Osteocalcin (OCN), a marker of osteoblast activity, has been implicated in the regulation of energy metabolism by the skeleton and thus may affect body fat measures. We examined the relationships of OCN to body fat measures and whether they vary according to markers of energy and vitamin D metabolism. Data was obtained from 58 obese adolescents aged 13–17.9 years (38 females, 8 black or African-American). We calculated total fat mass (FM) [dual X-ray absorptiometry (DXA)] and visceral adipose tissue (VAT) [computerized axial tomography (CT)]. Blood tests included leptin, OCN, 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH), thyroid function tests, and triglycerides. Markers of glucose metabolism were obtained from fasting and OGTT samples. Adolescents with 25(OH)D<20 ng/mL were considered deficient (n=17/58); none had high PTH (PTH≥65 pg/mL). OCN was associated with lower VAT (−84.27±33.89 mm2) and BMI (−0.10±0.05 kg/m2), not FM (p=0.597) in a core model including age, sex, race, geographic latitude, summer, height z-score, and tanner stage. Adding 25(OH)D deficiency and PTH attenuated the inverse association of OCN to VAT. We found a significant interaction of OCN and 25(OH)D deficiency on FM (0.37±0.18 kg, p=0.041) and BMI (0.28±0.10 kg/m2, p=0.007) in this adjusted model, which was further explained by leptin. Adding A1C to the core model modified the relationship of OCN to VAT (−93.08±35.05 mm2, p=0.011), which was further explained by HOMA-IR. In summary, these findings provide evidence for a relationship between OCN and body fat measures that is dependent on energy metabolism and vitamin D status among obese adolescents.