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1.
Br J Cancer ; 116(2): 227-236, 2017 Jan 17.
Article in English | MEDLINE | ID: mdl-28006818

ABSTRACT

BACKGROUND: Recent evidence suggests that bone-related parameters are the main prognostic factors for overall survival in advanced prostate cancer (PCa), with elevated circulating levels of alkaline phosphatase (ALP) thought to reflect the dysregulated bone formation accompanying distant metastases. We have identified that PCa cells express ALPL, the gene that encodes for tissue nonspecific ALP, and hypothesised that tumour-derived ALPL may contribute to disease progression. METHODS: Functional effects of ALPL inhibition were investigated in metastatic PCa cell lines. ALPL gene expression was analysed from published PCa data sets, and correlated with disease-free survival and metastasis. RESULTS: ALPL expression was increased in PCa cells from metastatic sites. A reduction in tumour-derived ALPL expression or ALP activity increased cell death, mesenchymal-to-epithelial transition and reduced migration. Alkaline phosphatase activity was decreased by the EMT repressor Snail. In men with PCa, tumour-derived ALPL correlated with EMT markers, and high ALPL expression was associated with a significant reduction in disease-free survival. CONCLUSIONS: Our studies reveal the function of tumour-derived ALPL in regulating cell death and epithelial plasticity, and demonstrate a strong association between ALPL expression in PCa cells and metastasis or disease-free survival, thus identifying tumour-derived ALPL as a major contributor to the pathogenesis of PCa progression.


Subject(s)
Alkaline Phosphatase/physiology , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/therapy , Alkaline Phosphatase/genetics , Animals , Cell Death/genetics , Cell Movement/genetics , Cells, Cultured , Disease Progression , Disease-Free Survival , HEK293 Cells , Humans , Male , Mice , Neoplasm Metastasis , Prostatic Neoplasms/genetics
2.
Biofabrication ; 8(3): 035001, 2016 07 19.
Article in English | MEDLINE | ID: mdl-27431399

ABSTRACT

Spinal fusion is the gold standard surgical procedure for degenerative spinal conditions when conservative therapies have been unsuccessful in rehabilitation of patients. Novel strategies are required to improve biocompatibility and osseointegration of traditionally used materials for lumbar cages. Furthermore, new design and technologies are needed to bridge the gap due to the shortage of optimal implant sizes to fill the intervertebral disc defect. Within this context, additive manufacturing technology presents an excellent opportunity to fabricate ergonomic shape medical implants. The goal of this study is to design and manufacture a 3D-printed lumbar cage for lumbar interbody fusion. Optimisations of the proposed implant design and its printing parameters were achieved via in silico analysis. The final construct was characterised via scanning electron microscopy, contact angle, x-ray micro computed tomography (µCT), atomic force microscopy, and compressive test. Preliminary in vitro cell culture tests such as morphological assessment and metabolic activities were performed to access biocompatibility of 3D-printed constructs. Results of in silico analysis provided a useful platform to test preliminary cage design and to find an optimal value of filling density for 3D printing process. Surface characterisation confirmed a uniform coating of nHAp with nanoscale topography. Mechanical evaluation showed mechanical properties of final cage design similar to that of trabecular bone. Preliminary cell culture results showed promising results in terms of cell growth and activity confirming biocompatibility of constructs. Thus for the first time, design optimisation based on computational and experimental analysis combined with the 3D-printing technique for intervertebral fusion cage has been reported in a single study. 3D-printing is a promising technique for medical applications and this study paves the way for future development of customised implants in spinal surgical applications.


Subject(s)
Biocompatible Materials , Printing, Three-Dimensional , Prostheses and Implants , Spinal Fusion/instrumentation , Stress, Mechanical , Adipose Tissue/cytology , Compressive Strength , Computer Simulation , Humans , Microscopy, Atomic Force , Models, Biological , Stem Cells/physiology
3.
Bone ; 81: 459-467, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26314515

ABSTRACT

Some anti-diabetic therapies can have adverse effects on bone health and increase fracture risk. In this study, we tested the skeletal effects of chronic administration of two Glucagon-like peptide-1 receptor agonists (GLP-1RA), increasingly used for type 2 diabetes treatment, in a model of osteoporosis associated bone loss and examined the expression and activation of GLP-1R in bone cells. Mice were ovariectomised (OVX) to induce bone loss and four weeks later they were treated with Liraglutide (LIR) 0.3mg/kg/day, Exenatide (Ex-4) 10 µg/kg/day or saline for four weeks. Mice were injected with calcein and alizarin red prior to euthanasia, to label bone-mineralising surfaces. Tibial micro-architecture was determined by micro-CT and bone formation and resorption parameters measured by histomorphometric analysis. Serum was collected to measure calcitonin and sclerostin levels, inhibitors of bone resorption and formation, respectively. GLP-1R mRNA and protein expression were evaluated in the bone, bone marrow and bone cells using RT-PCR and immunohistochemistry. Primary osteoclasts and osteoblasts were cultured to evaluate the effect of GLP-1RA on bone resorption and formation in vitro. GLP-1RA significantly increased trabecular bone mass, connectivity and structure parameters but had no effect on cortical bone. There was no effect of GLP-1RA on bone formation in vivo but an increase in osteoclast number and osteoclast surfaces was observed with Ex-4. GLP-1R was expressed in bone marrow cells, primary osteoclasts and osteoblasts and in late osteocytic cell line. Both Ex-4 and LIR stimulated osteoclastic differentiation in vitro but slightly reduced the area resorbed per osteoclast. They had no effect on bone nodule formation in vitro. Serum calcitonin levels were increased and sclerostin levels decreased by Ex-4 but not by LIR. Thus, GLP-1RA can have beneficial effects on bone and the expression of GLP-1R in bone cells may imply that these effects are exerted directly on the tissue.


Subject(s)
Bone and Bones/drug effects , Glucagon-Like Peptide-1 Receptor/agonists , Liraglutide/administration & dosage , Osteogenesis/drug effects , Osteoporosis/drug therapy , Peptides/administration & dosage , Venoms/administration & dosage , Adaptor Proteins, Signal Transducing , Animals , Bone Resorption , Bone and Bones/diagnostic imaging , Bone and Bones/metabolism , Calcitonin/blood , Diabetes Mellitus, Type 2/drug therapy , Disease Models, Animal , Exenatide , Female , Glycoproteins/blood , Glycoproteins/metabolism , Immunohistochemistry , Intercellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BL , Osteoblasts/cytology , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteocytes/cytology , Ovariectomy , RNA, Messenger/metabolism , Tibia/diagnostic imaging , X-Ray Microtomography
5.
Osteoporos Int ; 25(10): 2477-84, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25048011

ABSTRACT

SUMMARY: The basic mechanisms by which strontium ranelate acts on bone are still unclear. We show that an important action of strontium salts is to block calcification in cultures of osteoblasts, the bone-forming cells. These results suggest that strontium treatment could have previously overlooked effects on bone. INTRODUCTION: The basic mechanisms of action of strontium ranelate (SrR) on bone have remained unclear. We studied the direct actions of Sr(2+) salts in functional cultures of osteoblasts and osteoclasts. METHODS: Cultures of primary osteoblasts from rat calvariae and osteoclast-forming mouse marrow cells were treated continuously with either SrR or strontium chloride (SrCl2). RESULTS: Abundant, discretely mineralised 'trabecular' bone structures formed in control osteoblast cultures after 14 days. SrR at 0.01, 0.1 and 1 mM inhibited mineralisation to 59, 98 and 100 % (all p < 0.001) of control values, respectively. SrCl2 at the same concentrations caused similar inhibitions. Osteoblast cell numbers and alkaline phosphatase activity were unaltered. SrR dose-dependently reduced the formation of multinucleated osteoclasts from marrow mononuclear cells cultured on dentine for 8 days in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL), with a 50 % inhibition occurring at 1 mM; SrCl2 was slightly less effective, eliciting a maximal 30 % inhibition. Corresponding decreases in total resorption pit formation were observed, suggesting Sr(2+) salts affect osteoclast formation rather than resorptive activity. CONCLUSION: Our findings are consistent with the documented physicochemical inhibitory action of Sr(2+) on mineralisation but contrast with reports that Sr(2+) increases osteoblast activity and number in vitro. Our results suggest that rather than acting as an agent that 'uncouples' bone formation and resorption, Sr(2+) acts as a global inhibitor of bone cell function, with particularly marked effects on mineralisation. The potential effects of long-term Sr(2+) on secondary mineralisation in bone deserve investigation.


Subject(s)
Bone Density Conservation Agents/pharmacology , Calcification, Physiologic/drug effects , Osteoblasts/drug effects , Osteoclasts/drug effects , Thiophenes/pharmacology , Alkaline Phosphatase/metabolism , Animals , Cell Count , Cells, Cultured , Dose-Response Relationship, Drug , Osteoblasts/physiology , Osteoclasts/enzymology , Rats, Sprague-Dawley , Strontium/pharmacology , Thiophenes/administration & dosage
6.
Cell Death Differ ; 19(2): 194-208, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22048155

ABSTRACT

Signaling downstream of receptor tyrosine kinases controls cell differentiation and survival. How signals from different receptors are integrated is, however, still poorly understood. In this work, we have identified Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin repeat-rich membrane spanning) as a main player in the modulation of neurotrophin and vascular endothelial growth factor (VEGF) signaling in vivo, and a primary determinant for neuronal and cardiovascular development. Kidins220(-/-) embryos die at late stages of gestation, and show extensive cell death in the central and peripheral nervous systems. Primary neurons from Kidins220(-/-) mice exhibit reduced responsiveness to brain-derived neurotrophic factor, in terms of activation of mitogen-activated protein kinase signaling, neurite outgrowth and potentiation of excitatory postsynaptic currents. In addition, mice lacking Kidins220 display striking cardiovascular abnormalities, possibly due to impaired VEGF signaling. In support of this hypothesis, we demonstrate that Kidins220 constitutively interacts with VEGFR2. These findings, together with the data presented in the accompanying paper, indicate that Kidins220 mediates the integration of several growth factor receptor pathways during development, and mediates the activation of distinct downstream cascades according to the location and timing of stimulation.


Subject(s)
Membrane Proteins/metabolism , Nerve Growth Factors/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Blood Vessels/drug effects , Blood Vessels/growth & development , Blood Vessels/metabolism , Brain/blood supply , Brain/drug effects , Brain/enzymology , Brain-Derived Neurotrophic Factor/pharmacology , Caspase 3/metabolism , Cell Proliferation/drug effects , Excitatory Postsynaptic Potentials/drug effects , Membrane Proteins/deficiency , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Neurons/drug effects , Motor Neurons/metabolism , Nervous System/drug effects , Nervous System/metabolism , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , Signal Transduction/drug effects
7.
Exp Cell Res ; 312(10): 1693-702, 2006 Jun 10.
Article in English | MEDLINE | ID: mdl-16529738

ABSTRACT

We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO2 from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO2 was reduced further to 0.2%, bone nodule formation was almost abolished. The inhibitory effect of hypoxia on bone formation was partly due to decreased osteoblast proliferation, as measured by 3H-thymidine incorporation. Hypoxia also sharply reduced osteoblast alkaline phosphatase (ALP) activity and expression of mRNAs for ALP and osteocalcin, suggesting inhibition of differentiation to the osteogenic phenotype. Hypoxia did not increase the apoptosis of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O2 were less organized and much less abundant than in 20% O2 cultures. Furthermore, collagen produced by hypoxic osteoblasts contained a lower percentage of hydroxylysine residues and exhibited an increased sensitivity to pepsin degradation. These data demonstrate the absolute oxygen requirement of osteoblasts for successful bone formation and emphasize the importance of the vasculature in maintaining bone health. We recently showed that hypoxia also acts in a reciprocal manner as a powerful stimulator of osteoclast formation. Considered together, our results help to explain the bone loss that occurs at the sites of fracture, tumors, inflammation and infection, and in individuals with vascular disease or anemia.


Subject(s)
Cell Differentiation/physiology , Hypoxia , Osteoblasts/physiology , Osteogenesis/physiology , Oxygen/metabolism , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Animals, Newborn , Biomarkers/metabolism , Cells, Cultured , Collagen/metabolism , Collagen/ultrastructure , Osteoblasts/cytology , Rats , Rats, Sprague-Dawley
8.
Calcif Tissue Int ; 77(3): 167-74, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16075362

ABSTRACT

The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.


Subject(s)
Acidosis/metabolism , Calcification, Physiologic/physiology , Extracellular Matrix/metabolism , Osteoblasts/metabolism , Osteogenesis/physiology , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Animals, Newborn , Apoptosis , Cell Proliferation , Cells, Cultured , Collagen/metabolism , Extracellular Matrix Proteins , Gene Expression Regulation , Hydrogen-Ion Concentration , Nucleosomes/metabolism , Osteoblasts/pathology , Osteocalcin/genetics , Osteocalcin/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Skull/cytology
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