Spinor extended Lorentz-force like equations as consequence of a spinorial structure of space-time

As previously shown, the special relativistic dynamical equation of the Lorentz force type can be regarded as a consequence of a succession of space-time dependent infinitesimal Lorentz boosts and rotations. This insight indicate that the Lorentz-Force-like equation has a fundamental meaning in physics. We show how this result may be spinorially obtained starting out out from the application of an infinitesimal element of SL(2,C) to the individual spinors, which are regarded here as being more fundamental objects than four-vectors. In this way we get a set of new dynamical spinor equations inducing the extended Lorentz-Force-like equation in the Minkowski space-time and geometrically obtain the spinor form of the electromagnetic field tensor. The term extended refers to the dynamics of some additional degrees of freedom that may be associated with the intrinsic spin, namely, with the dynamics of three spacelike mutually orthogonal four-vectors, all of them orthogonal to the linear four-momentum of the object under consideration that finally, in the particle's proper frame. are identified with the generators of SU(2).Comment: 14 page

Nucleotides as regulators of bone cell function and mineralisation

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

Acidosis Is a Key Regulator of Osteoblast Ecto-Nucleotidase Pyrophosphatase/Phosphodiesterase 1 (NPP1) Expression and Activity

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 < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5‐fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions

Strontium potently inhibits mineralisation in bone-forming primary rat osteoblast cultures and reduces numbers of osteoclasts in mouse marrow cultures

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

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

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

Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

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