Extracellular pH: a fundamental regulator of bone cell function.

Systemic acidosis is associated with bone loss and impaired bone mineralisation. The aim of this PhD project was to further investigate the action of extracellular pH on the function of osteoclasts and osteoblasts. I showed that blood-derived human osteoclasts exhibited a highly reproducible acid-activation response, with maximal activation close to pH 7.0, and little activity at blood pH (7.4). These experiments also provided strong evidence that accessory cells, such as osteoblasts or stromal cells, are not required for acid-activation of resorption. The pH-activation profile of human osteoclasts was similar to that of the recently discovered KT-sensing human G- protein-coupled receptor OGR1. Expression of OGR1 and TDAG8 (another GPCR) was detected in human osteoclasts and was upregulated by low pH. I obtained evidence that the multifunctional receptor TRPV1, which senses protons, heat and capsaicin, was expressed by human osteoclasts and was also upregulated at pH 7.0. Moreover, I showed that the alkaloid capsaicin strongly stimulated osteoclasts in non- acidified conditions. To date, only pertussis toxin has been reported to activate osteoclasts without co-stimulation by H. Using mouse bone organ cultures I found that resorption-associated factors TRACP, cathepsin K and TRAF-6 were also upregulated by acidosis. The effect of PTH on human osteoclasts was also studied. I showed that PTH directly stimulates human osteoclasts in the absence of osteoblasts, but only when acid-activated. This finding suggests that the dogma that PTH stimulation of osteoclast is osteoblast-mediated may not be correct. Studies using primary rat osteoblast cultures showed that the formation of mineralised bone nodules is inhibited by acidosis. The same pH reduction, which increases Ca2+ and PO43" solubility of hydroxyapatite by 2- and 4-fold respectively, did not alter collagen production or osteoblast proliferation but decreased alkaline phosphatase activity and expression. Thus, the primary effect of acidosis on osteoblast function is to cause a selective inhibition of bone mineralisation. In conclusion, this study showed that the important "double negative" action of acidosis on bone cells is consistent with a pathophysiological role of bone as a reserve of base to buffer excess protons when the kidneys and lungs are unable to maintain acid-base balance within narrow physiological limits

The P2X7 Receptor is an Important Regulator of Extracellular ATP Levels

Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signaling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells), possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05–0.5 pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are fourfold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1–100 μM AZ10606120, A438079, and KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079, and KN-62, at 0.1–10 μM, decreased ATP release by mature osteoclasts by up to 70, 60, and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM or brefeldin A) had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1–10 μM) also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signaling in bone

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

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 <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation

Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

AbstractAllopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro

Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

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

Simulated interventions to ameliorate age-related bone loss indicate the importance of timing

Bone remodeling is the continuous process of bone resorption by osteoclasts and bone formation by osteoblasts, in order to maintain homeostasis. The activity of osteoclasts and osteoblasts is regulated by a network of signaling pathways, including Wnt, parathyroid hormone (PTH), RANKL/OPG and TGF-β, in response to stimuli such as mechanical loading. During aging there is a gradual loss of bone mass due to dysregulation of signaling pathways. This may be due to a decline in physical activity with age and/or changes in hormones and other signaling molecules. In particular, hormones such as PTH have a circadian rhythm which may be disrupted in aging. Due to the complexity of the molecular and cellular networks involved in bone remodeling, several mathematical models have been proposed to aid understanding of the processes involved. However, to date there are no models which explicitly consider the effects of mechanical loading, the circadian rhythm of PTH and the dynamics of signaling molecules on bone remodeling. Therefore, we have constructed a network model of the system using a modular approach which will allow further modifications as required in future research. The model was used to simulate the effects of mechanical loading and also the effects of different interventions such as continuous or intermittent administration of PTH. Our model predicts that the absence of regular mechanical loading and/or an impaired PTH circadian rhythm leads to a gradual decrease in bone mass over time which can be restored by simulated interventions and that the effectiveness of some interventions may depend on their timing

The effect of bone marrow microenvironment on the functional properties of the therapeutic bone marrow-derived cells in patients with acute myocardial infarction

<p>Abstract</p> <p>Background</p> <p>Treatment of acute myocardial infarction with stem cell transplantation has achieved beneficial effects in many clinical trials. The bone marrow microenvironment of ST-elevation myocardial infarction (STEMI) patients has never been studied even though myocardial infarction is known to cause an imbalance in the acid-base status of these patients. The aim of this study was to assess if the blood gas levels in the bone marrow of STEMI patients affect the characteristics of the bone marrow cells (BMCs) and, furthermore, do they influence the change in cardiac function after autologous BMC transplantation. The arterial, venous and bone marrow blood gas concentrations were also compared.</p> <p>Methods</p> <p>Blood gas analysis of the bone marrow aspirate and peripheral blood was performed for 27 STEMI patients receiving autologous stem cell therapy after percutaneous coronary intervention. Cells from the bone marrow aspirate were further cultured and the bone marrow mesenchymal stem cell (MSC) proliferation rate was determined by MTT assay and the MSC osteogenic differentiation capacity by alkaline phosphatase (ALP) activity assay. All the patients underwent a 2D-echocardiography at baseline and 4 months after STEMI.</p> <p>Results</p> <p>As expected, the levels of pO₂, pCO₂, base excess and HCO₃were similar in venous blood and bone marrow. Surprisingly, bone marrow showed significantly lower pH and Na⁺and elevated K⁺levels compared to arterial and venous blood. There was a positive correlation between the bone marrow pCO₂and HCO₃levels and MSC osteogenic differentiation capacity. In contrast, bone marrow pCO₂and HCO₃levels displayed a negative correlation with the proliferation rate of MSCs. Patients with the HCO₃level below the median value exhibited a more marked change in LVEF after BMC treatment than patients with HCO₃level above the median (11.13 ± 8.07% vs. 2.67 ± 11.89%, P = 0.014).</p> <p>Conclusions</p> <p>Low bone marrow pCO₂and HCO₃levels may represent the optimal environment for BMCs in terms of their efficacy in autologous stem cell therapy in STEMI patients.</p

Serum amyloid A inhibits RANKL-induced osteoclast formation

When mouse bone marrow-derived macrophages were stimulated with serum amyloid A (SAA), which is a major acute-phase protein, there was strong inhibition of osteoclast formation induced by the receptor activator of nuclear factor kappaB ligand. SAA not only markedly blocked the expression of several osteoclast-associated genes (TNF receptor-associated factor 6 and osteoclast-associated receptor) but also strongly induced the expression of negative regulators (MafB and interferon regulatory factor 8). Moreover, SAA decreased c-fms expression on the cell surface via shedding of the c-fms extracellular domain. SAA also restrained the fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by the well-known SAA receptors (formyl peptide receptor 2, Toll-like receptor 2 (TLR2) or TLR4). These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis and suggest that SAA is an important endogenous modulator that regulates bone homeostasis.open