Incidence of bone and osteoid tissue formation in different animals (A–D) and percentage of bone area in pores of dogs (E) at 6, 12 and 24 after implantation.

<p>Osteoid formation in rats was not observed at 6 weeks in any of the ceramics, while at 12 and 24 weeks, only BCP ceramics showed signs of osteoid formation (A). In rabbits, all ceramics showed ectopic osteoid formation at all time points, with a highest incidence at 12 weeks. At all time points, BCP1100 showed a higher osteoid incidence than BCP1200, which was again higher than osteoid incidence in HA1200 (B). In dogs, osteoid formation was observed in all ceramics at all time points, except in HA1200 at 6 weeks. Like in rabbits, a decreasing osteoid incidence was observed from BCP11 to BCP1200 to HA1200 (C). All ceramic showed ectopic bone formation in dogs at all time points (D) following a trend that was similar to that of osteoid incidence (C). Quantification of % of ectopic bone formation in available pore area showed that, in general, BCP1100 showed higher values than BCP1200 and HA1200; however, these differences were not statistically significant (E).</p

XRD patterns of the three ceramics.

<p>The patterns of BCP1100 and BCP1200 demonstrated biphasic nature of the ceramics consisting of HA and β-TCP. No apparent differences were found as a result of difference in sintering temperature. The pattern of HA1200 was typical of phase-pure hydroxyapatite.</p

A summary of tissue response to the three types of ceramics in different animals.

<p>A summary of tissue response to the three types of ceramics in different animals.</p

Histological evaluation of tissue formation in the three ceramics upon intramuscular implantation in rats, rabbits and dogs.

<p>Representative images showing tissue response to BCP1100 (A, D, G), BCP1200 (B, E, H) and HA1200 (C, F, I) upon implantation in the femoral muscle of rat, dorsal muscle of rabbit and dog for 6 weeks. Decalcified sections, HE Staining, magnification = ×100, scale bar = 200 µm; V→ Blood vessel, CT→Connective tissue, M→Material, O→Osteoid tissue, B→Bone.</p

Histological evaluation of bone formation upon implantation of the three ceramics in dorsal muscles of dogs for 12 weeks.

<p>The highest amount of osteoid and ectopic bone were observed in BCP1100 (A), followed by BCP1200 (B) an HA1200 (C). In all cases, bone was trabecular in appearance, with laminar bone and osteocytes contained in the lacunae, and infiltrated by blood vessels. Decalcified sections, Masson’s trichrome staining, magnification = ×100, scale bar = 200 µm; V→Blood vessel, CT→Connective tissue, M→Material, O→Osteoid tissue, B→Bone.</p

SEM micrographs showing macrostructure (A–C) and surface microstructure (D–F) of BCP1100 (A,D), BCP1200 (B,E) and HA1200 (C,F).

<p>All ceramics were porous with similar macroporous structure (A–C). The surface of the BCP1100 (D) ceramic exhibited smaller grains and a larger number of micropores than the chemically identical BCP1200, that was sintered at 1200°C (E). Grain size and number of micropores in HA1200 were between those of BCP110 and BCP1200 (F), which is in accordance with specific weight measurements in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107044#pone-0107044-t001" target="_blank">Table 1</a>. Scale bar = 1 mm for A–C and 10 µm for D–F.</p

Physico-chemical characterization of biphasic calcium phosphate sintered at 1100°C (BCP1100), of biphasic calcium phosphate sintered at 1200°C (BCP1200) and of phase-pure hydroxyapatite sintered at 1200°C (HA1200).

<p>Physico-chemical characterization of biphasic calcium phosphate sintered at 1100°C (BCP1100), of biphasic calcium phosphate sintered at 1200°C (BCP1200) and of phase-pure hydroxyapatite sintered at 1200°C (HA1200).</p

Dose-dependent effects of curcumin on bladder cancer cell proliferation.

<p>T-24 (2a) and AY-27 (2b) cells were plated in 12-well plates (0.8×10⁵ cells/well) and cultured for 48 hr (40–50% confluence) as described in Methods. Following exposure to various concentrations of curcumin for 24 h, cells were washed with PBS and incubated for another 24 h in curcumin-free medium. Cells incubated with DMSO (0.1%) alone were treated as controls. DMSO alone had little impact on tumor cell growth (data not shown). Cell proliferation was assessed on the basis of cell viability, measured by MTT assay. Cell viability was expressed as percent of viability observed in DMSO-treated control cells. Values are from a representative (of two) (2a) or 3–8 (mean ± S.E) determinations (2b). ^*P<0.05, ^**P<0.01,^***P<0.005.</p

Effect of Ki-67-7 and curcumin on the regulatory proteins of cell cycle phases and apoptosis: Western blot analysis.

<p>Bladder cancer cells (T-24 and AY-27) were cultured and exposed to Ki-67-7 and curcumin as described above in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048567#pone-0048567-g003" target="_blank">Figures 3A and 3B</a>. At the end of the treatment period, total protein was extracted and subjected to Western blotting using appropriate antibodies as described in Methods. For purposes of associating proteins with specific roles, they were grouped in separate categories, such as gene transcription (A), cell-cycle progression (B) and apoptosis (C). β-actin was used as the internal control. Since different proteins (e.g. Cyclin D1 and NF-κB) from the same membrane were identified in separate categories, the same β-actin blot is displayed on more than one occasion. Data presented are western blots from a single experiment, which is representative of 2–3 identical experiments.</p

Molecular targets of CusiRNA.

<p>The scheme displays the possible molecular targets of Ki-67-7 and curcumin when used in combination (CusiRNA). Arrowheads indicate the activation of indicated proteins; hammer heads indicate inhibition and the broken lines represent the possible role for putative intermediates. Further details are provided in the text.</p