ß,γ-Methylene-ATP and its metabolite medronic acid affect both arterial media calcification and bone mineralization in non-CKD and CKD rats.

Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PPi/Pi ratio). Also, it has been shown that ATP analogs (ie, ß,γ-methylene-ATP [ß,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of ß,γ-meATP (2 mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that ß,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4 mg/kg ß,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of ß,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of Enpp1 and Enpp3 was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.

Pathological calcification in canine tendon-derived cells is modulated by extracellular ATP.

Tendon calcification is a commonly associated with degenerative tendinopathy of the Achilles tendons in dogs. It is characterised by the formation of calcific deposits and is refractory to treatment, often re-forming after surgical removal. Little is known about its pathogenesis and therefore the aims of this study were to develop an in vitro model of canine tendon calcification and use this model to investigate mechanisms driving calcification. Cells from the canine Achilles tendon were cultured with different calcifying media to establish which conditions were best able to induce specific, cell-mediated calcification. Once optimum calcification conditions had been established, the effect of ATP treatment on calcification was assessed. Results revealed that 2 mM di-sodium phosphate combined with 2 mM calcium chloride provided the optimum calcifying conditions, increasing calcium deposition and expression of osteogenic-related genes similar to those observed in tendon calcification in vivo. ATP treatment inhibited calcification in a dose-dependent manner, reducing calcium deposition and increasing cell viability, while osteogenic-related genes were no longer upregulated. In conclusion, the in vitro model of canine tendon calcification developed in this study provides the ability to study mechanisms driving tendon calcification, demonstrating that ATP plays a role in modulating tendon calcification that should be explored further in future studies.

Stable Sulforaphane Targets the Early Stages of Osteoclast Formation to Engender a Lasting Functional Blockade of Osteoclastogenesis.

Sulforaphane, the native but unstable form of SFX-01, is an antioxidant that activates the NRF2 and inhibits the NF-KB pathways to achieve its actions. Resolving the mechanism(s) by which SFX-01 serves to control the various osteoclastogenic stages may expose pathways that could be explored for therapeutic use. Here we seek to identify the stage of osteoclastogenesis targeted by SFX-01 and explore whether, like SFN, it exerts its actions via the NRF2 and NF-KB pathways. Osteoclasts generated from the bone marrow (BM) of mice were cultured with SFX-01 at different timepoints to examine each phase of osteoclastogenesis separately. This showed that SFX-01 exerted actions throughout the process of osteoclastogenesis, but had its largest effects in the early osteoclast precursor differentiation stage. Thus, treatment with SFX-01 for the duration of culture, for the initial 3 days differentiation or for as little as the first 24 h was sufficient for effective inhibition. This aligned with data suggesting that SFX-01 reduced DC-STAMP levels, osteoclast nuclear number and modified cytoskeletal architecture. Pharmacological regulation of the NRF2 pathways, via selective inhibitors/activators, supported the anti-osteoclastogenic roles of an SFX-01-mediated by NRF2 activation, as well as the need for tight NF-KB pathway regulation in osteoclast formation/function.

A Machine Learning-Based Image Segmentation Method to Quantify In Vitro Osteoclast Culture Endpoints.

Quantification of in vitro osteoclast cultures (e.g. cell number) often relies on manual counting methods. These approaches are labour intensive, time consuming and result in substantial inter- and intra-user variability. This study aimed to develop and validate an automated workflow to robustly quantify in vitro osteoclast cultures. Using ilastik, a machine learning-based image analysis software, images of tartrate resistant acid phosphatase-stained mouse osteoclasts cultured on dentine discs were used to train the ilastik-based algorithm. Assessment of algorithm training showed that osteoclast numbers strongly correlated between manual- and automatically quantified values (r = 0.87). Osteoclasts were consistently faithfully segmented by the model when visually compared to the original reflective light images. The ability of this method to detect changes in osteoclast number in response to different treatments was validated using zoledronate, ticagrelor, and co-culture with MCF7 breast cancer cells. Manual and automated counting methods detected a 70% reduction (p < 0.05) in osteoclast number, when cultured with 10 nM zoledronate and a dose-dependent decrease with 1-10 µM ticagrelor (p < 0.05). Co-culture with MCF7 cells increased osteoclast number by ≥ 50% irrespective of quantification method. Overall, an automated image segmentation and analysis workflow, which consistently and sensitively identified in vitro osteoclasts, was developed. Advantages of this workflow are (1) significantly reduction in user variability of endpoint measurements (93%) and analysis time (80%); (2) detection of osteoclasts cultured on different substrates from different species; and (3) easy to use and freely available to use along with tutorial resources.

Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PPi) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PPi decreased osteoclast formation and resorptive activity by ≤50 %. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PPi present for the entire (0-21 days) or latter stages of culture (7-21/14-21 days) decreased bone mineralisation by ≤95 %. However, PPi present for the differentiation phase only (0-7/0-14 days) increased bone formation (≤70 %). Prolonged treatment with PPi resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PPi (≤3-fold). PPi levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PPi reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35 %) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PPi. ATP release from osteoclasts and osteoblasts was decreased ≤60 % by PPi and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gαi subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PPi. The actions of PPi on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PPi, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PPi to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PPi levels and respond accordingly. Our data also raise the possibility that some actions of PPi on bone cells could be mediated by a Gαi-linked GPCR.

Deletion of the P2Y2 receptor aggravates internal elastic lamina calcification in chronic kidney disease mice through upregulation of alkaline phosphatase and lipocalin-2.

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y2 receptor. This study investigates whether deletion of the P2Y2 receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n = 8), (ii) P2Y2 R-/- mice with normal renal function (n = 8), (iii) wild type mice with CKD (n = 27), and (iv) P2Y2 R-/- mice with CKD (n = 22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7 weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y2 receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median ± IQR), CKD-wild type: 0.34 ± 4.3 mg calcium/g wet tissue and CKD-P2Y2 R-/- : 4.0 ± 13.2 mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y2 R-/- mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y2 receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.

Modulation of osteoblast differentiation and function by the P2X4 receptor.

Bone cells are known to express multiple P2 receptor subtypes, and the functional effects of receptor activation have been described for many of these. One exception is the P2X4 receptor, which despite strong expression in osteoblasts and osteoclasts, has no defined functional activity. This study used the selective P2X4 receptor antagonists, 5-BDBD and PSB-12062, to investigate the role of this receptor in bone. Both antagonists (≥ 0.1 µM) dose-dependently decreased bone formation by 60-100%. This was accompanied by a ≤ 70% decrease in alkaline phosphatase activity, a ≤ 40% reduction in cell number, and a ≤ 80% increase in the number of adipocytes present in the culture. The analysis of gene expression showed that levels of osteoblast marker genes (e.g. Alpl, Bglap) were decreased in 5-BDBD treated cells. Conversely, expression of the adipogenic transcription factor PPARG was increased 10-fold. In osteoclasts, high doses of both antagonists were associated with a reduction in osteoclast formation and resorptive activity by ≤ 95% and ≤ 90%, respectively. Taken together, these data suggest that the P2X4 receptor plays a role in modulating bone cell function. In particular, it appears to influence osteoblast differentiation favouring the osteogenic lineage over the adipogenic lineage.

High bone mass in mice can be linked to lower osteoclast formation, resorptive capacity, and restricted in vitro sensitivity to inhibition by stable sulforaphane.

Mouse strains can have divergent basal bone mass, yet this phenotype is seldom reflected in the design of studies seeking to identify new modulators of bone resorption by osteoclasts. Sulforaphane exerts inhibitory effects on in vitro osteoclastogenesis in cells from C57BL/6 mice. Here, we explore whether a divergent basal bone mass in different mouse strains is linked both to in vitro osteoclastogenic potential and to SFX-01 sensitivity. Accordingly, osteoclasts isolated from the bone marrow (BM) of C57BL/6, STR/Ort and CBA mice with low, high, and intermediate bone mass, respectively, were cultured under conditions to promote osteoclast differentiation and resorption; they were also treated with chemically stabilised sulforaphane (SFX-01) and respective sensitivity to inhibition evaluated by counting osteoclast number/resorption activity on dentine discs. We observed that osteoclastogenesis exhibited different macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-Β ligand sensitivity in these mouse strains, with cells from C57BL/6 and CBA generating higher osteoclast numbers than STR/Ort; the latter formed only half as many mature osteoclasts. We found that 100 nM SFX-01 exerted a potent and significant reduction in osteoclast number and resorptive activity in cells derived from C57BL/6 mice. In contrast, 10-fold higher SFX-01 concentrations were required for similar inhibition in CBA-derived cells and, strikingly, a further 2.5-fold greater concentration was required for significant restriction of osteoclast formation/function in STR/Ort. These data are consistent with the notion that the BM osteoclast precursor population contributes to the relative differences in mouse bone mass and that mice with higher bone mass exhibit lower in vitro osteoclastogenic potential as well as reduced sensitivity to inhibition by SFX-01.

N-acetylcysteine (NAC) differentially affects arterial medial calcification and bone formation: The role of l-cysteine and hydrogen sulphide.

Arterial medial calcification (AMC) is the deposition of calcium phosphate in the arteries. AMC is widely thought to share similarities with physiological bone formation; however, emerging evidence suggests several key differences between these processes. N-acetylcysteine (NAC) displays antioxidant properties and can generate hydrogen sulphide (H2 S) and glutathione (GSH) from its deacetylation to l-cysteine. This study found that NAC exerts divergent effects in vitro, increasing osteoblast differentiation and bone formation by up to 5.5-fold but reducing vascular smooth muscle cell (VSMC) calcification and cell death by up to 80%. In vivo, NAC reduced AMC in a site-specific manner by 25% but had no effect on the bone. The actions of l-cysteine and H2 S mimicked those of NAC; however, the effects of H2 S were much less efficacious than NAC and l-cysteine. Pharmacological inhibition of H2 S-generating enzymes did not alter the actions of NAC or l-cysteine; endogenous production of H2 S was also unaffected. In contrast, NAC and l-cysteine increased GSH levels in calcifying VSMCs and osteoblasts by up to 3-fold. This suggests that the beneficial actions of NAC are likely to be mediated via the breakdown of l-cysteine and the subsequent GSH generation. Together, these data show that while the molecular mechanisms driving the actions of NAC appear similar, the downstream effects on cell function differ significantly between osteoblasts and calcifying VSMCs. The ability of NAC to exert these differential actions further supports the notion that there are differences between the development of pathological AMC and physiological bone formation. NAC could represent a therapeutic option for treating AMC without exerting negative effects on bone.

Regulation of mineralisation in bone and vascular tissue: a comparative review.

Biomineralisation, the deposition of mineral onto a matrix, can be both a physiological and pathological process. Bone formation involves the secretion of an extracellular matrix (ECM) by osteoblasts and subsequent mineralisation of that matrix. It is regulated by a number of local and systemic factors and is necessary for maintenance of normal bone health. Conversely, mineralisation (or calcification) of soft tissues, including the vasculature, is detrimental to that tissue, leading to diseases such as arterial medial calcification (AMC). The mechanisms underlying AMC development are not fully defined, though it is thought that vascular smooth muscle cells (VSMCs) drive this complex, cell-mediated process. Similarly, AMC is regulated by a variety of enzymes and molecules, many of which have already been implicated in the regulation of bone mineralisation. This review will provide an overview of the similar, and sometimes opposing effects of these signalling molecules on the regulation of bone mineralisation and AMC.

2-Oxothiazolidine-4-carboxylic acid inhibits vascular calcification via induction of glutathione synthesis.

Arterial medial calcification (AMC), the deposition of hydroxyapatite in the medial layer of the arteries, is a known risk factor for cardiovascular events. Oxidative stress is a known inducer of AMC and endogenous antioxidants, such as glutathione (GSH), may prevent calcification. GSH synthesis, however, can be limited by cysteine levels. Therefore, we assessed the effects of the cysteine prodrug 2-oxothiazolidine-4-carboxylic acid (OTC), on vascular smooth muscle cell (VSMC) calcification to ascertain its therapeutic potential. Human aortic VSMCs were cultured in basal or mineralising medium (1 mM calcium chloride/sodium phosphate) and treated with OTC (1-5 mM) for 7 days. Cell-based assays and western blot analysis were performed to assess cell differentiation and function. OTC inhibited calcification ≤90%, which was associated with increased ectonucleotide pyrophosphatase/phosphodiesterase activity, and reduced apoptosis. In calcifying cells, OTC downregulated protein expression of osteoblast markers (Runt-related transcription factor 2 and osteopontin), while maintaining expression of VSMC markers (smooth muscle protein 22α and α-smooth muscle actin). GSH levels were significantly reduced by 90% in VSMCs cultured in calcifying conditions, which was associated with declines in expression of gamma-glutamylcysteine synthetase and GSH synthetase. Treatment of calcifying cells with OTC blocked the reduction in expression of both enzymes and prevented the decline in GSH. This study shows OTC to be a potent and effective inhibitor of in vitro VSMC calcification. It appears to maintain GSH synthesis which may, in turn, prevent apoptosis and VSMCs gaining osteoblast-like characteristics. These findings may be of clinical relevance and raise the possibility that treatment with OTC could benefit patients susceptible to AMC.

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

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 < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Extracellular Nucleotides Regulate Arterial Calcification by Activating Both Independent and Dependent Purinergic Receptor Signaling Pathways.

Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5'-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor-pyrophosphate and the calcification stimulator-inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization.

Extracellular pyrophosphate: The body's "water softener".

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.

Inhibition of vascular smooth muscle cell calcification by ATP analogues.

Arterial medial calcification (AMC) has been associated with phenotypic changes in vascular smooth muscle cells (VSMCs) that reportedly makes them more osteoblast-like. Previous work has shown that ATP/UTP can inhibit AMC directly via P2 receptors and indirectly by NPP1-mediated hydrolysis to produce the mineralisation inhibitor, pyrophosphate (PPi). This study investigated the role of P2X receptors in the inhibitory effects of extracellular nucleotides on VSMC calcification. We found that Bz-ATP, α,ß-meATP and ß,γ-meATP inhibited calcification by up to 100%. Culture in a high-phosphate medium (2 mM) was associated with increased VSMC death and apoptosis; treatment with Bz-ATP, α,ß-meATP and ß,γ-meATP reduced apoptosis to levels seen in non-calcifying cells. Calcification was also associated with alterations in the protein levels of VSMC (e.g. SM22α and SMA) and osteoblast-associated (e.g. Runx2 and osteopontin) markers; Bz-ATP, α,ß-meATP and ß,γ-meATP attenuated these changes in protein expression. Long-term culture with Bz-ATP, α,ß-meATP and ß,γ-meATP resulted in lower extracellular ATP levels and an increased rate of ATP breakdown. P2X receptor antagonists failed to prevent the inhibitory effects of these analogues suggesting that they act via P2X receptor-independent mechanisms. In agreement, the breakdown products of α,ß-meATP and ß,γ-meATP (α,ß-meADP and methylene diphosphonate, respectively) also dose-dependently inhibited VSMC calcification. Furthermore, the actions of Bz-ATP, α,ß-meATP and ß,γ-meATP were unchanged in VSMCs isolated from NPP1-knockout mice, suggesting that the functional effects of these compounds do not involve NPP1-mediated generation of PPi. Together, these results indicate that the inhibitory effects of ATP analogues on VSMC calcification and apoptosis in vitro may be mediated, at least in part, by mechanisms that are independent of purinergic signalling and PPi.

The in vitro behaviour of canine osteoblasts derived from different bone types.

BACKGROUND: Our understanding of the biology of osteoblasts is important as they underpin bone remodelling, fracture healing and processes such as osseointegration. Osteoblasts isolated from human humeral samples display distinctive biological activity in vitro, which relates to the samples' bone types (subchondral (S), trabecular (T), cortical (C)). Our aim was to isolate primary osteoblast cultures from different bone types from the proximal femur of a clinical population of dogs presented for total hip replacement and compare the behaviour of the osteoblasts derived from different bone types, to identify a preferred bone type for isolation. RESULTS: No differences were found for osteoblast doubling time (median for S = 2.9, T = 3.1 and C = 2.71 days, respectively; p = 0.33), final cell number (median for S = 54,849, T = 49,733, C = 61,390 cells/cm2; p = 0.34) or basal tissue non-specific alkaline phosphatase (TNAP) activity (median for S = 0.02, T = 0.02, C = 0.03 U/min/mg protein; p = 0.81) between bone types after 6 days of culture in basal media. There were no differences in mineralizing TNAP activity (S = 0.02, T = 0.02, C = 0.03 U/min/mg protein, p = 0.84) or in mineralized area (S = 0.05, T = 0.04, C = 0.04%, p = 0.92) among cells from different bone types. CONCLUSIONS: There is no significant difference in mean doubling time, basal or mineralizing TNAP activity or mineralized area in osteoblasts derived from subchondral, cortical, or trabecular bone types from the canine femoral head. However, there appears to be a high level of inter-animal variability in the studied parameters, which was independent of age, body mass, and sex. Trabecular isolate osteoblasts have the least variation of the bone types studied, and therefore should be considered a preferred source for primary osteoblast cultures. The work here provides baselines for canine osteoblast function, which has utility for future comparative studies.

Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts.

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 ß-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.

Isolation and Generation of Osteoblasts.

This chapter describes the isolation, culture, and staining of osteoblasts. The key advantages of this assay are that it allows direct measurement of bone matrix deposition and mineralization, as well as yielding good quantities of osteoblasts at defined stages of differentiation for molecular and histological analysis. An additional focus of this chapter will be the culture of osteoblasts from less conventional animal species.

Spina bifida-predisposing heterozygous mutations in Planar Cell Polarity genes and Zic2 reduce bone mass in young mice.

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.