Co-cultured OCLs gene expression analysis.

<p>Relative quantification (2^-ΔΔCt) of gene expression after 7 days of OCLs co-cultured with OBs grown in direct contact with all the tested samples. The graphs show the average and standard error of the technical triplicate of Oscar, Itgβ3 and CtsK, respect to the expression of the cells only, used as a control. Statistical significant difference among the samples is indicated in the graph: **p≤0.01.</p

OBs viability and apoptosis.

<p>(A) shows the percentage of live OBs respect to the total cells counted and (B) shows the percentage of apoptotic OBs respect to the total cells counted. Mean and standard error (n = 3) represented as the percentage of the total counted cells, after 7 and 14 days of culture in direct contact with all the tested samples. Statistical significant differences among the samples are indicated in both graphs: *p≤0.05. (C) Different examples of nuclear fragmentation in OBs stained with DAPI are indicated with red arrows. Scale bar 50 μm.</p

OBs gene expression analysis.

<p>(a) and (c) Relative quantification (2^-ΔΔCt) of gene expression after 7 and (b) and (d) 14 days of OBs cultured in direct contact with all the tested samples. The graph shows the average and standard error of the technical triplicate of Osterix and IBSP, respect to the expression of the cells only, used as a control. Statistical significant differences among the samples are indicated in the graphs: *p≤0.05 and ***p≤0.001.</p

OCLs gene expression analysis.

<p>Relative quantification (2^-ΔΔCt) of gene expression after 7 days of OCLs cultured in direct contact with all the tested samples. Average and standard error of the technical triplicate of Oscar, Itgβ3 and CtsK, respect to the expression of the cells only, were indicated. Statistical significant difference among the samples is indicated in the graph: *p≤0.05.</p

Samples identification.

<p>Labelling of the samples, weight percentage (wt%) of LF loaded onto HA and LF and HA concentration (μg/ml) of the samples tested in the study.</p><p>Samples identification.</p

OPG/RANKL ratio in OBs/OCLs co-culture.

<p>In the graph is reported the ratio of the soluble factors measured by ELISA kit. Mean and standard error of three replicates are shown. Statistical significant differences among the samples are indicated in the graph: *p≤0.05.</p

Synthesis and Preliminary <i>in Vivo</i> Evaluation of Well-Dispersed Biomimetic Nanocrystalline Apatites Labeled with Positron Emission Tomographic Imaging Agents

In recent years, biomimetic synthetic apatite nanoparticles (AP-NPs), having chemical similarity with the mineral phase of bone, have attracted a great interest in nanomedicine as potential drug carriers. To evaluate the therapeutic perspectives of AP-NPs through the mechanisms of action and organs they interact with, the noninvasive monitoring of their <i>in vivo</i> behavior is of paramount importance. To this aim, here the feasibility to radiolabel AP-NPs (“naked” and surface-modified with citrate to reduce their aggregation) with two positron emission tomographic (PET) imaging agents ([¹⁸F]­NaF and ⁶⁸Ga-NO2AP^BP) was investigated. [¹⁸F]­NaF was used for the direct incorporation of the radioisotope into the crystal lattice, while the labeling by surface functionalization was accomplished by using ⁶⁸Ga-NO2AP^BP (a new radio-metal chelating agent). The labeling results with both tracers were fast, straightforward, and reproducible. AP-NPs demonstrated excellent ability to bind relevant quantities of both radiotracers and good <i>in vitro</i> stability in clinically relevant media after the labeling. <i>In vivo</i> PET studies in healthy Wistar rats established that the radiolabeled AP-NPs gave significant PET signals and they were stable over the investigated time (90 min) since any tracer desorption was detected. These preliminary <i>in vivo</i> studies furthermore showed a clear ability of citrated versus naked AP-NPs to accumulate in different organs. Interestingly, contrary to naked AP-NPs, citrated ones, which unveiled higher colloidal stability in aqueous suspensions, were able to escape the first physiological filter, i.e., the lungs, being then accumulated in the liver and, to a lesser extent, in the spleen. The results of this work, along with the fact that AP-NPs can be also functionalized with targeting ligands, with therapeutic agents, and also with metals for a combination of different imaging modalities, make AP-NPs very encouraging materials for further investigations as theranostic agents in nanomedicine

Magnetic Bioinspired Hybrid Nanostructured Collagen–Hydroxyapatite Scaffolds Supporting Cell Proliferation and Tuning Regenerative Process

A bioinspired mineralization process was applied to develop biomimetic hybrid scaffolds made of (Fe²⁺/Fe³⁺)-doped hydroxyapatite nanocrystals nucleated on self-assembling collagen fibers and endowed with super-paramagnetic properties, minimizing the formation of potentially cytotoxic magnetic phases such as magnetite or other iron oxide phases. Magnetic composites were prepared at different temperatures, and the effect of this parameter on the reaction yield in terms of mineralization degree, morphology, degradation, and magnetization was investigated. The influence of scaffold properties on cells was evaluated by seeding human osteoblast-like cells on magnetic and nonmagnetic materials, and differences in terms of viability, adhesion, and proliferation were studied. The synthesis temperature affects mainly the chemical–physical features of the mineral phase of the composites influencing the degradation, the microstructure, and the magnetization values of the entire scaffold and its biological performance. In vitro investigations indicated the biocompatibility of the materials and that the magnetization of the super-paramagnetic scaffolds, induced applying an external static magnetic field, improved cell proliferation in comparison to the nonmagnetic scaffold

pH-Responsive Delivery of Doxorubicin from Citrate–Apatite Nanocrystals with Tailored Carbonate Content

In this work, the efficiency of bioinspired citrate-functionalized nanocrystalline apatites as nanocarriers for delivery of doxorubicin (DOXO) has been assessed. The nanoparticles were synthesized by thermal decomplexing of metastable calcium/citrate/phosphate solutions both in the absence (Ap) and in the presence (cAp) of carbonate ions. The presence of citrate and carbonate ions in the solution allowed us to tailor the size, shape, carbonate content, and surface chemistry of the nanoparticles. The drug-loading efficiency of the two types of apatite was evaluated by means of the adsorption isotherms, which were found to fit a Langmuir–Freundlich behavior. A model describing the interaction between apatite surface and DOXO is proposed from adsorption isotherms and ζ-potential measurements. DOXO is adsorbed as a dimer by means of a positively charged amino group that electrostatically interacts with negatively charged surface groups of nanoparticles. The drug-release profiles were explored at pHs 7.4 and 5.0, mimicking the physiological pH in the blood circulation and the more acidic pH in the endosome-lysosome intracellular compartment, respectively. After 7 days at pH 7.4, cAp-DOXO released around 42% less drug than Ap-DOXO. However, at acidic pH, both nanoassemblies released similar amounts of DOXO. <i>In vitro</i> assays analyzed by confocal microscopy showed that both drug-loaded apatites were internalized within GTL-16 human carcinoma cells and could release DOXO, which accumulated in the nucleus in short times and exerted cytotoxic activity with the same efficiency. cAp are thus expected to be a more promising nanocarrier for experiments <i>in vivo</i>, in situations where intravenous injection of nanoparticles are required to reach the targeted tumor, after circulating in the bloodstream

Figures S1 - S4 and Table S1 from A combined low-frequency electromagnetic and fluidic stimulation for a controlled drug release from superparamagnetic calcium phosphate nanoparticles: potential application for cardiovascular diseases

Figure S1: Transmission Electron Microscopy (TEM) images of FeHAs (a, b) at two different magnifications and HAs (c); Figure S2: Hydrodynamic diameter distributions of FeHA and HA in HEPES buffer 0.1 M, pH 7.4.; Table S1: Technical data of each component of the MEBD; Figure S3: Adsorption kinetics of IBU on (▪) FeHAs and (▫) HAs.; Figure S4: Adsorption isotherms of IBU on (▪) FeHAs and (▫) HAs. Separate points are the experimental data; dotted lines indicate Sips fits of isotherm data