Inorganic pyrophosphate (PP i ) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP i are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP i on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP i . This inhibition could be reversed by Tnap, but not if PP i was bound to mineral. PP i also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP i was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP i , hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP i inhibits Tnap-mediated P i release from ␤-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP i prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

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Ultrafine surface features are commonly used to modulate the cellular activity of a variety of materials including ceramics, [1,2] composites, [3,4] nanofibers, [5,6] and polymers. [7,8] However, the main challenge for materials in contact with bone remains the development of a material with both favorable surface and bulk properties to modulate not only cell-substrate interactions but also to ensure the long-term stability of the implant. This challenge has motivated researchers to develop bulk nanostructured materials while applying different surface treatments to improve cellular adhesion and metabolic activities. Here, in a combined approach involving materials science and cell and molecular biology, the responses of pre-osteoblast and fibroblast cell lines to novel nanostructured titanium substrates produced by high-pressure torsion (HPT) is assessed and compared with the cellular activity on coarse-grained, annealed titanium substrates. The degree of osteoblast attachment is notably increased on the HPT-processed titanium substrates. The improved cellular response is attributed to the nanostructured features of the samples, which are characterized by an ultrafine grain size (< 50 nm), and a high degree of surface wettability associated with a distinctive oxide layer formed on the surface. This finding provides a valuable advantage for HPT-processed titanium over conventional and coated titanium implants, as both the mechanical and physical properties, along with biological activities, are improved. The most developed severe plastic deformation (SPD) techniques for producing bulk nonporous samples are equal-channel angular pressing (ECAP) and HPT. [9,10] Recent studies indicate that high pressure during SPD significantly affects the development of the crystal structure and consequently enables the production of materials composed of sub-micrometer-or nanometer-sized metal crystals, both on the surface and in the bulk, that have reduced porosity, cracks, and other macroscopic defects. [11,12] These characteristics make the material very attractive for advanced applications in the aerospace, sport, transportation, and, in particular, medical industries.[13] Along with nanostructured surface features, material processed by HPT has a good combination of high strength and high ductility at room temperature. These are two desirable but rarely co-existing properties important for the longterm stability of metallic implants. [14,15] Although many reports are available on the bulk characterization of these materials, reports detailing the effects of high pressure on the surface properties of nanostructured titanium are mostly fragmented, and its significance with regards to cell-substrate interactions has not been addressed. In this work, commercially pure titanium was used to fabricate substrates with different nano/microstructures through HPT and annealing processes. After fabrication of the substrates, surface characterization was performed by orientation image microscopy (OIM), transmission electron microscopy (T...

Quartz crystal microbalance with dissipation monitoring (QCM-D) is used for real-time in situ detection of cytoskeletal changes in live primary endothelial cells in response to different cytomorphic agents; namely, the surfactant Triton-X 100 (TX-100) and bacterial lipopolysaccharide (LPS). Reproducible dissipation versus frequency (Df) plots provide unique signatures of the interactions between endothelial cells and cytomorphic agents. While the QCM-D response for TX-100 can be described in two steps (changes in the osmotic pressure of the medium prior to observing the expected cell lysis), LPS results in a different single-phase signal. A complementary analysis is carried out to evaluate the possible competitive effects of TX-100 and LPS through the QCM-D response to BAEC stress by analyzing the Df plots obtained. Experiments with non-toxic components (fibronectin or serum) produce a different QCM-D response than that observed for the toxic chemicals, suggesting the use of Df plot signatures for the possible differentiation between cytotoxic or non-cytotoxic effects. Observations obtained by QCM-D signals are confirmed by conducting fluorescence microscopy at the same time. Our results show that a fast (few minutes) sensing response can be obtained in situ and in real-time. The conclusions from this study suggest that QCM-D can potentially be used in biodetection for applications in drug screening tests and diagnosis.

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