Ultrananocrystalline diamond coated implants for enhanced osseointegration

Osteoporosis causes bones to become weak and brittle. It is known that the alterations in bone metabolism associated to osteoporosis can impair bone healing around implants and affect their osseointegration. The main objective of this study was the development of new nanostructured implant materials based on ultrananocrystalline diamond (UNCD) coatings for enhancing osseointegration. The films were deposited on Ti substrates by microwave plasma CVD from 17% CH4/N2 gas mixtures and modified by O2 or NH3/N2 plasmas. The modifications rendered the H-terminated hydrophobic as-grown films hydrophilic. The interaction of endothelial (EA.hy926) and osteosarcoma (MG63) cells with differently modified UNCD surfaces was investigated by proteome analyses. It revealed the identification of over 19 000 proteins (extracellular and cytosolic). They correspond to 508 (Ti), 634 (UNCD), 651 (O-UNCD), and 668 (NH2-UNCD) protein groups. The interaction network analysis showed differences in the connectivity of inferred protein networks between the ECM niches, which suggests the presence of specific cell microenvironments on O- and NH2-terminated UNCD surfaces. Our results show that due to a favorable combination of surface chemical and topological properties the UNCD films, as-grown and especially after their plasma modifications, may serve as superior scaffolding for promoting the cell attachment and growth during osseointegration

Ultrananocrystalline diamond coated implants for enhanced osseointegration

Osteoporosis causes bones to become weak and brittle. It is known that the alterations in bone metabolism associated to osteoporosis can impair bone healing around implants and affect their osseointegration. The main objective of this study was the development of new nanostructured implant materials based on ultrananocrystalline diamond (UNCD) coatings for enhancing osseointegration. The films were deposited on Ti substrates by microwave plasma CVD from 17% CH4/N2 gas mixtures and modified by O2 or NH3/N2 plasmas. The modifications rendered the H-terminated hydrophobic as-grown films hydrophilic. The interaction of endothelial (EA.hy926) and osteosarcoma (MG63) cells with differently modified UNCD surfaces was investigated by proteome analyses. It revealed the identification of over 19 000 proteins (extracellular and cytosolic). They correspond to 508 (Ti), 634 (UNCD), 651 (O-UNCD), and 668 (NH2-UNCD) protein groups. The interaction network analysis showed differences in the connectivity of inferred protein networks between the ECM niches, which suggests the presence of specific cell microenvironments on O- and NH2-terminated UNCD surfaces. Our results show that due to a favorable combination of surface chemical and topological properties the UNCD films, as-grown and especially after their plasma modifications, may serve as superior scaffolding for promoting the cell attachment and growth during osseointegration

In vitro bioactivity of glass-ceramic/fibroin composites

Bioactive composite materials were prepared by mixing 20 wt.% of silk fibroin (SF) and 80 wt.% of glassceramics from CaO-SiO2-P2O5-MgO system. In vitro bioactivity of the prepared composites was evaluated in 1.5 simulated body fluid (1.5 SBF) in static conditions. The obtained samples before and after in vitro tests were characterized by X-ray diffraction (XRD) analysis, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). The changes in 1.5 SBF solutions after soaking the samples were evaluated by inductively coupled plasma atomic emission spectroscopy (ICP-AES). MG63 osteosarcoma cells were used for the biological experiments. The obtained experimental data proved that the synthesized composites exhibit excellent in vitro bioactivity

W-7 inhibits H₂O₂-mediated Src activation in SK-BR-3 cells.

<p>(<i>A</i>) SK-BR-3 cells were incubated in the absence (-) and presence (+) of the indicated concentrations of W-12 or W-7 during 15 min. Thereafter, the cells were stimulated with 1 mM H₂O₂ for 15 min. The reaction was arrested and subjected to Western blot analysis as described in Materials and Methods, and probed with anti-phospho-Src (Y416), and anti-Src (total) and anti-GAPDH antibodies as loading controls. (<i>B</i>) The plot presents the mean ± SEM (n = 5) H₂O₂-dependent activation of Src in the absence (<i>None</i>) and presence of either 50 μM W-12 or 50 μM W-7 from experiments similar to those shown in <i>A</i>. Statistically significant differences with p < 0.05 (*) using the Student’s t-test are indicated.</p

Calmodulin directly interacts with Src in the absence and presence of Ca²⁺.

<p>(<i>A</i>) Ca²⁺-dependent calmodulin-affinity chromatography of Src solubilized from the membrane fraction of A431 cells was performed as described in Materials and Methods. The material loaded in the CaM-Sepharose column (<i>input</i>), the last fraction after washing with the Ca²⁺-buffer (<i>Ca</i>^<i>2+</i><i>-wash</i>), and the EGTA-eluted fractions were analyzed by Western blot using an anti-Src antibody. (<i>B</i>) Pull-down of Src solubilized from a membrane fraction of A431 cells was performed in the absence (-) and presence (+) of Ca²⁺ using CaM-Sepharose (<i>CaM-beads</i>) as described in Materials and Methods. Naked Sepharose 4B beads were used as negative binding control. The presence of bound Src to the beads was analyzed by Western blot using an anti-Src antibody. (<i>C</i>) Src was solubilized from a membrane fraction of A431 cells (2 mg protein) and immunoprecipitated (IP) using an anti-Src antibody as described in Materials and Methods. The immunocomplex was processed by Western blot using anti-CaM and anti-Src antibodies. Non-relevant rabbit IgG (~22 μg) was used as a negative control (mock IP).</p

Phospho-(Y)-mimetic CaM mutants also activate c-Src.

<p>(<i>A</i>) Auto-phosphorylation assays of recombinant c-Src (0.1 μg) was performed in a medium containing 15 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM dithiothreitol, 1 mM EGTA and where indicated 1.2 mM CaCl₂ (200 μM free Ca²⁺) in the presence of either wild type CaM or CaM(Y99D/Y138D) in a total volume of 100 μl. The samples were processed for SDS-PAGE and Western blot using anti-phospho-tyrosine and anti-Src antibodies as described in Materials and Methods. (<i>B</i>, <i>C</i>) The plots present the mean ± SEM (n = 2) c-Src phosphorylation in the presence of wild type CaM or CaM(Y99D/Y138D) in the absence and presence of free Ca²⁺ from experiments similar to the ones shown in <i>A</i>. (<i>D</i>) Auto-phosphorylation assays of recombinant c-Src (0.1 μg) was performed in a medium containing 15 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM dithiothreitol, 1 mM EGTA and where indicated 1.2 mM CaCl₂ (200 μM free Ca²⁺) in the absence or presence of either CaM(Y99D/Y138D) or CaM(Y99E/Y138E) in a total volume of 100 μl. The samples were processed for SDS-PAGE and Western blot using anti-phospho-tyrosine and anti-Src antibodies as described in Materials and Methods (<i>E</i>, <i>F</i>). The plots present the mean ± SEM c-Src phosphorylation in the presence of CaM(Y99D/Y138D) (n = 1) or CaM(Y99E/Y138E) (n = 2), and in the absence and presence of free Ca²⁺ from experiments similar to the ones shown in <i>A</i>.</p

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.This work was funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación [grant number SAF2014-52048-R (to A.V.)]; the Consejería de Educación de la Comunidad de Madrid [grant number S2011/BMD-2349 (to A.V.)]; the CSIC program i-COOP+ 2014 [grant number COOPA20053 (to A.V.)] and the European Commission [grant number PITNGA-2011-289033 (to A.V.)]; the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program [grant number PITN-GA-2011-289033 (to S.R.S.)]; the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela [grant number CDCH-UCV 03-00-6057-2005 (to V.S.)]; and [grant number PG-03-8728-2013 (to G.B.)]; and the Fondo Nacional de Ciencia, Tecnología e Innovación [grant number P-2011000884 (to G.B.)

Phosphorylation of different CaM species by c-Src.

<p>The different CaM species (2 μg) were assayed for phosphorylation by recombinant c-Src as described in Materials and Methods. The samples were probed with an anti-phospho-tyrosine antibody to detect the tyrosine-phosphorylated CaM species (<i>P-(Y)-CaM</i>) and auto-phosphorylated c-Src (<i>P-(Y)-Src</i>). The membranes were striped and reprobed with anti-CaM and anti-Src antibodies as loading controls.</p

Absorption spectra of the different CaM species.

<p>The plots show UV-light absorption spectra of recombinant wild type (<i>wt</i>) and the indicated Y/D (<i>panel A</i>) and Y/E (<i>panel B</i>) CaM mutants (2 mg/ml) purified as described in Materials and Methods and dialyzed against 20 mM Tris-HCl (pH 7.5).</p