Electron micrograph of poly(P) cells on the 3rd day of culture.

<p>A. Nucleolus (arrows) is smaller. Vacuoles seen outside of cytoplasm (arrowhead). B. A well-developed Golgi apparatus (G) can be seen. Coated pits (arrows) and swollen RER (asterisk) are frequently observed. A bundle of microfilaments (double arrow) can also be seen. C. Mitochondria (M) large and elongated. D. Low density cytoplasm with many vacuoles containing small granules and myelin-like structures (arrowhead). E. RER well developed, elongated and swollen at one end (asterisk). A coated pit (arrow) and a granule-like structure with an amorphous material (arrowhead) can be seen. (Bar = 0.5 µm).</p

Morphological change in poly(P)-treated cells.

<p>Phase contrast light microscopy. (Original magnification, ×200) A. MC3T3-E1 cells on 3rd day of culture. Cytoplasm extended like a rugby ball. B. MC3T3-E1 cells treated with poly(P) on the 3rd day of culture. Cells are thicker and the cytoplasm irregularly extended with vacuoles.</p

Effect of poly(P) on localization of collagen type I and osteopontin.

<p>A. Immunofluorescence using anti-collagen type I antibody on the 9th day of culture. (Original magnification, ×200 in a and b, ×400 in c and d) (a) Control cells. Peripheral area of the cytoplasm weakly positive. (b) Poly(P)-treated cells. Immunofluorescence intensity stronger than that of the control. Positive granular structures along the cytoplasm can be seen. (c) Higher magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086834#pone-0086834-g002" target="_blank">Figure 2a</a>. (d) Higher magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086834#pone-0086834-g002" target="_blank">Figure 2b</a>. B. Immunofluorescence using anti-osteopontin antibody. (Original magnification, ×200) (a) Control cells. Reaction is weakly positive. (b) Poly(P)-treated cells. Area around the nucleus shows strong positive reaction.</p

Electron micrograph of control cells on the 3rd day of culture.

<p>A. Slightly condensed chromatin in the nucleus. Nucleolus clearly demarcated. B. Higher magnification of area shown by large arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086834#pone-0086834-g004" target="_blank">Figure 4A</a>. Rounded mitochondria (M) and well-developed Golgi apparatus (G) can be seen. A granule (arrowhead) containing dense material, small granular structure and filamentous structure can also be seen. C. Higher magnification of area shown by small arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086834#pone-0086834-g004" target="_blank">Figure 4A</a>. Nucleolus (N) and bundle of microfilaments (double arrow) can be seen. D. Cytoplasm of control cells. Granules containing several myelin-like structures (arrowheads) visible. Rounded mitochondria (M) and elongated rRERs (arrow) can be seen. Several RERs (asterisks) swollen at one end. (Bar = 0.5 µm).</p

The effect of α, α’-bipyridyl, a ROS inhibitor, on polyphosphatase activity of rh-TRAP.

<p>The [³²P]-poly(P)₄₀ (0.346 mM) was incubated with recombinant human TRAP (7.3 mU/mL) in 100 mM Na-acetate buffer (pH 5.5) containing 40 mM sodium tartrate in the presence or absence of α, α’-bipyridyl (5 mM) for the indicated time periods at 37°C. The degradation of [³²P]-poly(P) was analyzed by 20% PAGE.</p

Multifunctional Biomaterial Coating Based on Bio-Inspired Polyphosphate and Lysozyme Supramolecular Nanofilm

Current implant materials have widespread clinical applications together with some disadvantages, the majority of which are the ease with which infections are induced and difficulty in exhibiting biocompatibility. For the efficient improvement of their properties, the development of interface multifunctional modification in a simple, universal, and environmently benign approach becomes a critical challenge and has acquired the attention of numerous scientists. In this study, a lysozyme-polyphosphate composite coating was fabricated for titanium­(Ti)-based biomaterial to obtain a multifunctional surface. This coating was easily formed by sequentially soaking the substrate in reduced-lysozyme and polyphosphate solution. Such a composite coating has shown predominant antibacterial activity against Gram-negative bacteria (<i>E. coli</i>) and improved cell adhesion, proliferation, and differentiation, which are much better than those of the pure substrate. This facile modification endows the biomaterial with anti-infective and potential bone-regenerative performance for clinical applications of biomaterial implants

Degradation of poly(P) by Sf9 cell culture supernatant containing rh-TRAP.

<p>The [³²P]-poly(P)₄₀ (panel A) or [³²P]-poly(P) with an average chain length of 40, 300, or 750 residues (panel B) (0.346 mM) were incubated with Sf9 cell culture supernatant containing rh-TRAP (7.3 mU/mL) in 100 mM Na-acetate buffer (pH 5.5) with 40 mM sodium tartrate for the indicated time periods at 37°C. Degradation products were analyzed by 20% PAGE.</p

poly(P) reduces LPS-induced NO release in macrophages.

<p>The cells were pretreated with poly(P)₁₃₀ (100 µM) for 5 min and stimulated with LPS (100 ng/mL) for 24 h. NO released (measured as nitrite) was determined by the Griess reaction. Values are expressed as means ± SD of the percentage of nitrite compared with LPS alone from six independent experiments using different cell preparations. **<i>p</i><0.01, significantly different from basal. #<i>p</i><0.05, significantly different from LPS alone (ANOVA with Bonferroni’s post-test).</p

The effects of poly(P) on the viability of macrophages.

<p>The cells were pretreated with Pi, poly(P)₁₄, poly(P)₆₀, or poly(P)₁₃₀ (1 mM) for 5 min and stimulated with LPS (100 ng/mL) for 24 h. Cell viability was then determined using the WST-8 assay (A) and trypan blue exclusion assay (B). H₂O₂ (250 µM, 24 h) was used as a positive control. Values are expressed as means ± SD of the percentage of cell viability (A) or cell number (B) compared with basal from three independent experiments using different cell preparations.</p

A schematic model of poly(P) function in bone remodeling.

<p>TRAP, which is secreted from osteoclasts, degrades poly(P) into intermediate poly(P) and subsequently into Pi. Poly(P) inhibits osteoclastic bone resorption, while intermediate poly(P) promotes differentiation and calcification of osteoblasts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078612#pone.0078612-Kawazoe1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078612#pone.0078612-Kawazoe2" target="_blank">[15]</a>. Thus, poly(P) may regulate bone remodeling.</p