35 results on '"Orriss I"'
Search Results
2. Osteoblast‐specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase‐1 engenders insulin resistance in high‐fat diet fed mice
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Roberts, F L, Rashdan, N A, Phadwal, K, Markby, G R, Dillon, S, Zoll, J, Berger, J, Milne, E, Orriss, I R, Karsenty, G, Le Saux, O, Morton, N M, Farquharson, C, and MacRae, V E
- Subjects
musculoskeletal diseases - Abstract
Supraphysiological levels of the osteoblast‐enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase‐1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast‐specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6‐week‐old mice lacking osteoblast NPP1 expression (osteoblast‐specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p
- Published
- 2020
3. Extracellular pyrophosphate: The body's 'water softener'
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Orriss, I R
- Abstract
Extracellular pyrophosphate (ePPi) was first identified as a key endogenous inhibitor of mineralisation in the 1960's by Fleisch and colleagues. The main source of ePPi seems to be extracellular ATP which is continually released from cells in a controlled way. ATP is rapidly broken down by enzymes including ecto-nucleotide pyrophosphatase/phosphodiesterases to produce ePPi. The major function of ePPi is to directly inhibit hydroxyapatite formation and growth meaning that this simple molecule acts as the body's own “water softener”. However, studies have also shown that ePPi can influence gene expression and regulate its own production and breakdown. This review will summarise our current knowledge of ePPi metabolism and how it acts to prevent pathological soft tissue calcification and regulate physiological bone mineralisation.
- Published
- 2020
4. Acidosis Inhibits Bone Formation by Osteoblasts In Vitro by Preventing Mineralization
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Brandao-Burch, A., Utting, J. C., Orriss, I. R., and Arnett, T. R.
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- 2005
- Full Text
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5. Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts
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Patel, J J, Bourne, L E, Davies, B K, Arnett, T R, MacRae, V E, Wheeler-Jones, C P D, and Orriss, I R
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musculoskeletal diseases ,cardiovascular system ,musculoskeletal system - Abstract
Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, in the medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. This study used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely used in vitro models of AMC and bone formation. Significant differences were identified between osteoblasts and calcifying VSMCs. First, osteoblasts formed large mineralised bone nodules that were associated with widespread deposition of an extracellular collagenous matrix. In contrast, VSMCs formed small discrete regions of calcification that were not associated with collagen deposition and did not resemble bone. Second, calcifying VSMCs displayed a progressive reduction in cell viability over time (≤7-fold), with a 50% increase in apoptosis, whereas osteoblast and control VSMCs viability remained unchanged. Third, osteoblasts expressed high levels of alkaline phosphatase (TNAP) activity and TNAP inhibition reduced bone formation by to 90%. TNAP activity in calcifying VSMCs was ∼100-fold lower than that of bone-forming osteoblasts and cultures treated with β-glycerophosphate, a TNAP substrate, did not calcify. Furthermore, TNAP inhibition had no effect on VSMC calcification. Although, VSMC calcification was associated with increased mRNA expression of osteoblast-related genes (e.g. Runx2, osterix, osteocalcin, osteopontin), the relative expression of these genes was up to 40-fold lower in calcifying VSMCs versus bone-forming osteoblasts. In summary, calcifying VSMCs in vitro display some limited osteoblast-like characteristics but also differ in several key respects: 1) their inability to form collagen-containing bone; 2) their lack of reliance on TNAP to promote mineral deposition; and, 3) the deleterious effect of calcification on their viability.
- Published
- 2019
6. Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts
- Author
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Utting, J. C., Robins, S. P., Brandao-Burch, A., Orriss, I. R., Behar, J., and Arnett, T. R.
- Published
- 2006
- Full Text
- View/download PDF
7. Strontium and osteoblast function
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Wornham, D. P., Hajjawi, M. O., Orriss, I. R., and Arnett, T. R.
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- 2015
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8. Spina bifida-predisposing heterozygous mutations in Planar Cell Polarity genes and Zic2 reduce bone mass in young mice
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Orriss, I R, Lanham, S A, Savery, D, Greene, N D E, Stanier, P, Oreffo, R, Copp, A J, and Galea, G L
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congenital, hereditary, and neonatal diseases and abnormalities ,nervous system diseases - Abstract
Fractures are a common comorbidity in children with the neural tube defect (NTD) spina bifida. Mutations in the Wnt/planar cell polarity (PCP) pathway contribute to NTDs in humans and mice, but whether this pathway independently determines bone mass is poorly understood. Here, we first confirmed that core Wnt/PCP components are expressed in osteoblasts and osteoclasts in vitro. In vivo, we performed detailed µCT comparisons of bone structure in tibiae from young male mice heterozygous for NTD-associated mutations versus WT littermates. PCP signalling disruption caused by Vangl2 (Vangl2Lp/+) or Celsr1 (Celsr1Crsh/+) mutations significantly reduced trabecular bone mass and distal tibial cortical thickness. NTD-associated mutations in non-PCP transcription factors were also investigated. Pax3 mutation (Pax3Sp2H/+) had minimal effects on bone mass. Zic2 mutation (Zic2Ku/+) significantly altered the position of the tibia/fibula junction and diminished cortical bone in the proximal tibia. Beyond these genes, we bioinformatically documented the known extent of shared genetic networks between NTDs and bone properties. 46 genes involved in neural tube closure are annotated with bone-related ontologies. These findings document shared genetic networks between spina bifida risk and bone structure, including PCP components and Zic2. Genetic variants which predispose to spina bifida may therefore independently diminish bone mass.
- Published
- 2018
9. Metabolic properties of the osteoclast
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Arnett, T R and Orriss, I R
- Abstract
Osteoclasts are defined as cells capable of excavating 3-dimensional resorption pits in bone and other mineralised tissues. They are derived from the differentiation/fusion of promonocytic precursors, and are usually large, multinucleated cells. In common with other cells from this myeloid lineage such as macrophages and dendritic cells, they are adapted to function in hypoxic, acidic environments. The process of bone resorption is rapid and is presumably highly energy-intensive, since osteoclasts must actively extrude protons to dissolve hydroxyapatite mineral, whilst secreting cathepsin K to degrade collagen, as well as maintaining a high degree of motility. Osteoclasts are well known to contain abundant mitochondria but they are also able to rely on glycolytic (anaerobic) metabolism to generate the ATP needed to power their activity. Their primary extracellular energy source appears to be glucose. Excessive accumulation of mitochondrial reactive oxygen species in osteoclasts during extended periods of high activity in oxygen-poor environments may promote apoptosis and help to limit bone resorption — a trajectory that could be termed “live fast, die young”. In general, however, the metabolism of osteoclasts remains a poorly-investigated area, not least because of the technical challenges of studying actively resorbing cells in appropriate conditions.
- Published
- 2017
10. Inhibition of arterial medial calcification and bone mineralization by extracellular nucleotides: The same functional effect mediated by different cellular mechanisms
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Patel, J J, Zhu, D, Opdebeeck, B, D'Haese, P, Millán, J L, Bourne, L E, Wheeler-Jones, C P D, Arnett, T R, MacRae, V E, and Orriss, I R
- Abstract
Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast‐like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non‐calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100‐fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto‐nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi. In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.
- Published
- 2017
11. Activation of the P2Y2 receptor regulates bone cell function by enhancing ATP release
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Orriss, I R, Gueneri, D, Hajjawai, M O, Shaw, K, Patel, J J, and Arnett, T R
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Bone cells constitutively release ATP into the extracellular environment where it acts locally via P2 receptors to regulate bone cell function. Whilst P2Y2 receptor stimulation regulates bone mineralisation, the functional effects of this receptor in osteoclasts remain unknown. This investigation used the P2Y2 receptor knockout (P2Y2R−/−) mouse model to investigate the role of this receptor in bone. MicroCT analysis of P2Y2R−/− mice demonstrated age-related increases in trabecular bone volume (≤48%), number (≤30%) and thickness (≤17%). In vitro P2Y2R−/− osteoblasts displayed a 3-fold increase in bone formation and alkaline phosphatase activity, whilst P2Y2R−/− osteoclasts exhibited a 65% reduction in resorptive activity. Serum cross-linked C-telopeptide levels (CTX, resorption marker) were also decreased (≤35%). The resorption defect in P2Y2R−/− osteoclasts was rescued by the addition of exogenous ATP, suggesting that an ATP deficit could be a key factor in the reduced function of these cells. In agreement, we found that basal ATP release was reduced up to 53% in P2Y2R−/− osteoclasts. The P2Y2 receptor agonists, UTP and 2-thioUTP, increased osteoclast activity and ATP release in wild-type but not in P2Y2R−/− cells. This indicates that the P2Y2 receptor may regulate osteoclast function indirectly by promoting ATP release. UTP and 2-thioUTP also stimulate ATP release from osteoblasts suggesting that the P2Y2 receptor exerts a similar function in these cells. Taken together, our findings are consistent with the notion that the primary action of P2Y2 receptor signalling in bone is to regulate extracellular ATP levels.
- Published
- 2017
12. Musculoskeletal: Where are we with treating musculoskeletal disorders?
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Orriss, I R and MacRae, V E
- Published
- 2016
13. Pyrophosphate: a key inhibitor of mineralisation
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Orriss, I R, Arnett, T R, and Russell, R G G
- Published
- 2016
14. Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro
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Hajjawi, M O R, Patel, J J, Corcelli, M, Arnett, T R, and Orriss, I R
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musculoskeletal diseases - Abstract
Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30–50 % at a concentration of 1 μM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone.
- Published
- 2016
15. Acidosis Is a Key Regulator of Osteoblast Ecto-Nucleotidase Pyrophosphatase/Phosphodiesterase 1 (NPP1) Expression and Activity
- Author
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Orriss, I R, Key, M L, Hajjawi, M O R, Millan, J L, and Arnett, T R
- Abstract
Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1–6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P
- Published
- 2015
16. Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1-/- mice
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Hajjawi, M O R, MacRae, V E, Huesa, C, Boyde, A, Millan, J L, Arnett, T R, Orriss, I R, Queen Mary London, Roslin, Sanford-Burnham, and UCL
- Abstract
Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues.
- Published
- 2014
17. Tumour-derived alkaline phosphatase regulates tumour growth, epithelial plasticity and disease-free survival in metastatic prostate cancer.
- Author
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Rao, S R, Snaith, A E, Marino, D, Cheng, X, Lwin, S T, Orriss, I R, Hamdy, F C, and Edwards, C M
- 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. [ABSTRACT FROM AUTHOR]- Published
- 2017
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18. Fibrillin-1 expression, which regulates of TGF-B bioavailability, is modified during osteoarthritis and mutations lead to osteoarthritis
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Ramanayake, W., Jones, H., Orriss, I., Arnett, T., Pitsillides, A., Denton, C., Abraham, D., and Poulet, B.
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- 2014
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19. Kidins220/ARMS mediates the integration of the neurotrophin and VEGF pathways in the vascular and nervous systems.
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Cesca, F, Yabe, A, Spencer-Dene, B, Scholz-Starke, J, Medrihan, L, Maden, C H, Gerhardt, H, Orriss, I R, Baldelli, P, Al-Qatari, M, Koltzenburg, M, Adams, R H, Benfenati, F, and Schiavo, G
- Subjects
PROTEIN-tyrosine kinases ,CELL differentiation ,NEUROTROPHINS ,VASCULAR endothelial growth factors ,PREGNANCY - 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. [ABSTRACT FROM AUTHOR]- Published
- 2012
- Full Text
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20. Osteoblast-specific Enpp1 deficiency engenders increased bone mass and insulin resistance.
- Author
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Roberts, F. L., Rashdan, N., Orriss, I., Milne, E., Morton, N., Farquharson, C., and Macrae, V. E.
- Subjects
INSULIN resistance ,OSTEOBLASTS ,TYPE 2 diabetes ,MEDICAL sciences - Published
- 2019
21. Strontium potently inhibits mineralisation in bone-forming primary rat osteoblast cultures and reduces numbers of osteoclasts in mouse marrow cultures
- Author
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Wornham, D P, Hajjawi, M O R, Orriss, I R, Arnett, T R, and UCL
- Subjects
inorganic chemicals ,musculoskeletal diseases - Abstract
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.
22. The role of purinergic signalling in the musculoskeletal system
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Orriss, I R
23. The in vitro behaviour of canine osteoblasts derived from different bone types
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Meeson, R L, Perpétuo, I P, Parsons, K J, Orriss, I R, Shah, M, Pitsillides, A A, and Doube, M
24. The P2X7 receptor is an important regulator of extracellular ATP levels
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Brandao-Burch, A, Key, M L, Patel, J J, Arnett, T R, and Orriss, I R
- Subjects
musculoskeletal diseases
25. Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation
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Orriss, I R, Key, M L, Hajjawi, M O R, and Arnett, T R
- Abstract
Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to
26. Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation
- Author
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Orriss, I R, Arnett, T R, George, J, and Witham, M D
27. Design and fabrication of 3D-printed anatomically shaped lumbar cage for intervertebra disc (IVD) degeneration treatment
- Author
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Serra, T, Capelli, C, Toumpaniari, R, Orriss, I R, Leong, J, Dalgarno, K, and Kalaskar, D
- Subjects
ComputingMethodologies_DOCUMENTANDTEXTPROCESSING
28. Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats
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Orriss, I R, Hajjawi, M O R, Huesa, C, MacRae, V E, Arnett, T R, Royal (Dick) School of Veterinary Studies, and UCL
- Abstract
The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone‑forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14‑28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised ‘trabecular‑shaped’ bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21‑28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco's modified Eagle's medium (DMEM) after approximately 14 days (although ~3‑fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 µg/ml) for osteogenic differentiation and β‑glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well‑established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1‑100 µM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone‑forming rat osteoblasts.
29. Nucleotides as regulators of bone cell function and mineralisation
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Hajjawi, M. O. R., Arnett, T. R., and Orriss, I. R.
- Subjects
570 - Abstract
Most cells, including bone cells, release ATP into the extracellular environment. A considerable body of previous work has shown that ATP, acting through the P2 receptors, inhibits bone formation by osteoblasts and increases bone resorption by osteoclasts. This work focuses on the action of two key breakdown products of ATP, pyrophosphate and adenosine on bone cell function. Pyrophosphate, a ubiquitous physicochemical inhibitor of mineralisation, is formed from extracellular ATP by the action of ecto-nucleotide pyrophosphatase phosphodiesterases (NPPs); in bone these enzymes act in opposition to alkaline phosphatase. Adenosine, which can be generated in a number of ways from ATP, has been previously reported to stimulate both osteoblast and osteoclast function. However, using in vitro cultures, I found that it had little or no effect on the differentiation and bone forming capacity of rat osteoblasts, nor on the formation and resorptive function of mouse osteoclasts. I investigated the possibility that osteocytes, which form an interconnected cellular network within bone, might regulate mineralisation via NPPs. I found that cultured, primary osteocyte-like cells derived from mouse bone expressed Enpp1 mRNA. Osteocyte lacunae in the femora of Enpp1-/- mice imaged by scanning electron microscopy were found to be reduced in area by about 35%; indirect estimates of lacunar size using microCT imaging were in agreement. These results are consistent with the notion that ATP-derived pyrophosphate is important for maintenance of osteocyte lacunae size. Enpp1-/- mouse bones (humerus) were found to have reduced cortical bone diameter, reduced cortical porosity and an increased endosteal diameter compared to wild types, suggesting that the knockout phenotype also involves increased bone resorption and decreased bone formation. Histology and microCT of Enpp1-/- mice confirmed inappropriate joint mineralisation and showed that cartilage in the trachea and ear pinna was also mineralised, as were whisker sheaths. Osteoblasts, osteoclasts and osteocytes cultured in vitro from Enpp1-/- mice were found to release less ATP compared to cells from Enpp1+/+ mice in static conditions and after fluid flow stimulation. Enpp1-/- osteoblasts and osteoclasts also contained higher levels of intracellular ATP. Enpp1-/- osteoblasts showed increased bone production in vitro compared to Enpp1+/+; no effects of Enpp1 knockout on the formation or resorptive activity of osteoclasts were noted. Sclerostin, an osteocyte-derived inhibitor of WNT signalling and bone formation, was found to increase Enpp1 mRNA expression and NPP activity of osteoblasts, without affecting ALP in vitro. These results emphasise the importance of ATP and its breakdown product pyrophosphate in regulating mineralisation.
- Published
- 2014
30. Glomerulonephritis and autoimmune vasculitis are independent of P2RX7 but may depend on alternative inflammasome pathways.
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Prendecki M, McAdoo SP, Turner-Stokes T, Garcia-Diaz A, Orriss I, Woollard KJ, Behmoaras J, Cook HT, Unwin R, Pusey CD, Aitman TJ, and Tam FW
- Subjects
- Adenosine Triphosphate metabolism, Animals, Caspase 1 metabolism, Caspases, Inflammasomes metabolism, Inflammation metabolism, Inflammation pathology, Interleukin-1beta metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, Rats, Rats, Inbred WKY, Glomerulonephritis, Receptors, Purinergic P2X7 metabolism, Vasculitis metabolism, Vasculitis pathology
- Abstract
P2RX7, an ionotropic receptor for extracellular adenosine triphosphate (ATP), is expressed on immune cells, including macrophages, monocytes, and dendritic cells and is upregulated on nonimmune cells following injury. P2RX7 plays a role in many biological processes, including production of proinflammatory cytokines such as interleukin (IL)-1β via the canonical inflammasome pathway. P2RX7 has been shown to be important in inflammation and fibrosis and may also play a role in autoimmunity. We have developed and phenotyped a novel P2RX7 knockout (KO) inbred rat strain and, taking advantage of the human-resembling unique histopathological features of rat models of glomerulonephritis, we induced three models of disease: nephrotoxic nephritis, experimental autoimmune glomerulonephritis, and experimental autoimmune vasculitis. We found that deletion of P2RX7 does not protect rats from models of experimental glomerulonephritis or the development of autoimmunity. Notably, treatment with A-438079, a P2RX7 antagonist, was equally protective in WKY WT and P2RX7 KO rats, revealing its 'off-target' properties. We identified a novel ATP/P2RX7/K
+ efflux-independent and caspase-1/8-dependent pathway for the production of IL-1β in rat dendritic cells, which was absent in macrophages. Taken together, these results comprehensively establish that inflammation and autoimmunity in glomerulonephritis is independent of P2RX7 and reveals the off-target properties of drugs previously known as selective P2RX7 antagonists. Rat mononuclear phagocytes may be able to utilise an 'alternative inflammasome' pathway to produce IL-1β independently of P2RX7, which may account for the susceptibility of P2RX7 KO rats to inflammation and autoimmunity in glomerulonephritis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)- Published
- 2022
- Full Text
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31. Design and fabrication of 3D-printed anatomically shaped lumbar cage for intervertebral disc (IVD) degeneration treatment.
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Serra T, Capelli C, Toumpaniari R, Orriss IR, Leong JJ, Dalgarno K, and Kalaskar DM
- Subjects
- Adipose Tissue cytology, Compressive Strength, Computer Simulation, Humans, Microscopy, Atomic Force, Models, Biological, Stem Cells physiology, Biocompatible Materials, Printing, Three-Dimensional, Prostheses and Implants, Spinal Fusion instrumentation, Stress, Mechanical
- 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.
- Published
- 2016
- Full Text
- View/download PDF
32. Chronic administration of Glucagon-like peptide-1 receptor agonists improves trabecular bone mass and architecture in ovariectomised mice.
- Author
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Pereira M, Jeyabalan J, Jørgensen CS, Hopkinson M, Al-Jazzar A, Roux JP, Chavassieux P, Orriss IR, Cleasby ME, and Chenu C
- Subjects
- 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, 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
- 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
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33. Role of the P2Y13 receptor in the differentiation of bone marrow stromal cells into osteoblasts and adipocytes.
- Author
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Biver G, Wang N, Gartland A, Orriss I, Arnett TR, Boeynaems JM, and Robaye B
- Subjects
- Adipocytes metabolism, Animals, Cell Differentiation physiology, Female, Male, Mesenchymal Stem Cells cytology, Mice, Mice, Inbred C57BL, Mice, Knockout, Osteoblasts metabolism, Receptors, Purinergic P2 genetics, Receptors, Purinergic P2 metabolism, Adipocytes cytology, Mesenchymal Stem Cells metabolism, Osteoblasts cytology, Receptors, Purinergic P2 physiology
- Abstract
Accumulating evidence indicates that extracellular nucleotides, signaling through purinergic receptors, play a significant role in bone remodeling. Mesenchymal stem cells (MSCs) express functional P2Y receptors whose expression level is regulated during osteoblast or adipocyte differentiation. P2Y13 -deficient mice were previously shown to exhibit a decreased bone turnover associated with a reduction in the number of both osteoblasts and osteoclasts on the bone surfaces. We therefore examined whether P2Y13 R activation was involved in the osteogenic differentiation of MSC. Our study demonstrated that ADP stimulation of P2Y13 R(+/+) (but not P2Y13 R(-/-) ) adherent bone marrow stromal cells (BMSCs) increased significantly the formation of alkaline phosphatase-colony-forming units (CFU-ALP) as well as the expression of osteoblastic markers (osterix, alkaline phosphatase, and collagen I) involved in the maturation of preosteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13 R(-/-) BMSC and the level of osteoblastic gene expression after osteogenic stimulation were strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y13 R(-/-) BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y13 R(+/+) MSC. Interestingly, we observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y13 R(-/-) mice. In conclusion, our findings indicate that the P2Y13 R plays an important role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. Therefore, the P2Y13 receptor can be considered as a new pharmacological target for the treatment of bone diseases like osteoporosis. STEM Cells 2013;31:2747-2758., (© AlphaMed Press.)
- Published
- 2013
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34. Purinergic signaling in bone.
- Author
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Jørgensen NR, Adinolfi E, Orriss I, and Schwarz P
- Published
- 2013
- Full Text
- View/download PDF
35. Bone phenotypes of P2 receptor knockout mice.
- Author
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Orriss I, Syberg S, Wang N, Robaye B, Gartland A, Jorgensen N, Arnett T, and Boeynaems JM
- Subjects
- Animals, Bone Density genetics, Bone Resorption genetics, Extracellular Space metabolism, Mice, Mice, Knockout, Nucleotides metabolism, Bone and Bones abnormalities, Bone and Bones metabolism, Phenotype, Receptors, Purinergic P2 genetics, Signal Transduction genetics
- Abstract
The action of extracellular nucleotides is mediated by ionotropic P2X receptors and G-protein coupled P2Y receptors. The human genome contains 7 P2X and 8 P2Y receptor genes. Knockout mice strains are available for most of them. As their phenotypic analysis is progressing, bone abnormalities have been observed in an impressive number of these mice: distinct abnormalities in P2X7-/- mice, depending on the gene targeting construct and the genetic background, decreased bone mass in P2Y1-/- mice, increased bone mass in P2Y2-/- mice, decreased bone resorption in P2Y6-/- mice, decreased bone formation and bone resorption in P2Y13-/- mice. These findings demonstrate the unexpected importance of extracellular nucleotide signalling in the regulation of bone metabolism via multiple P2 receptors and distinct mechanisms involving both osteoblasts and osteoclasts.
- Published
- 2011
- Full Text
- View/download PDF
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