Synthesis of FeHA nanoparticles
FeHA nanoparticles were prepared according to the method described in our previous study . Briefly, a basic suspension of calcium hydroxide Ca(OH)2 (Aldrich, 95 wt% pure, 50 g in 400 ml of H2O) was stirred and heated up to 40°C. FeCl2·4H2O (Aldrich, ≥ 99 wt% pure, 12.74 g in 75 ml of H2O) and FeCl3·6H2O (Aldrich, 97 wt% pure, 17.86 g in 75 ml of H2O) solutions were contemporarily added to the basic suspension as sources of Fe2+ and Fe3+ ions. The total amounts of iron ions with respect to calcium ions were adjusted so as to obtain: Fe/Ca = 20 mol%.
Soon after a phosphoric acid (Aldrich, 85 wt% pure, 44.40 g in 300 ml of H2O) solution was drop-wise added into a basic suspension of calcium hydroxide containing iron ions, over a period of 2 h, under constant heating and stirring.
The reaction products were kept in suspension by constant stirring and heating for 1 h and then left ageing for 24 h at room temperature without further stirring. The precipitate was separated from mother liquor by centrifugation, then washed with distilled water and centrifuged three times; finally it was freeze-dried and sieved at 150 μm.
For the in vivo test, FeHA nanopowder was processed in order to obtain a more stable granulate (400-600 μm). In detail, the powder was hydrated with distilled water and agglomerated. Agglomerates were dried at 40°C for 48 h and sieved in the range 400-600 μm. Glass vials containing 0.5 g of granulates were prepared and sterilized with 25 γ-ray. The control group consisted of a bone filler made of HA granulate of identical size (400-600 μm), already commercially available, and stabilized for one hour at 300°C (FinGranule, Finceramica Faenza Spa, Italy).
FeHA nanoparticles characterization
FeHA phase composition was determined by X-ray powder diffraction (XRD), performed by a D8 Advance Diffractometer (Bruker, Karlsruhe, Germany) using CuKa radiation at 40 kV and 40 mA. XRD spectra were recorded in the 2 h range 10–60° or 15–120°, with a step size of 0.02 and a counting time of 1 s (corresponding to 185 s using a conventional detector). Quantitative evaluation of phase compositions and cell parameters was performed by full-profile Rietveld analysis of the XRD spectrum (TOPAS v4.2, Bruker AXS, Karlsruhe, Germany). Computer simulation of XRD patterns of FeHA powders based on structural models was carried out by the aid of the software Powder cell 2.4 (W. Krause & G. Nolze, 2000).
ICP-OES quantitative analysis made use of inductively coupled plasma – atomic emission spectrometry (ICP-AES: Liberty 200, Varian, Clayton South, Australia) to determine the overall content of Ca, P and Fe. Samples were previously prepared as follows: 20 mg of powder were dissolved in 2 ml of HNO3 (Aldrich, 65 wt% pure) and the solution volume was increased up to 100 ml with deionised water. The obtained values were expressed in terms of (Fe + Ca)/P mol, Ca/P mol and Fe/Ca molar %.
The analysis of powder morphology was carried out by high-resolution transmission electron microscopy (HRTEM) analyses, performed by a JEOL JEM 3010-UHR, operating at 300 kV.
Magnetic measurements were performed at higher field in a superconducting quantum interference device (SQUID) magnetometer from Quantum Design (San Diego, CA, USA), capable of operating from 1.8 to 350 K under a maximum applied magnetic field of H = 5 N A-1 m-1.
Saos-2 Human Osteoblast-like cells purchased from Lonza (Italy) were cultured in Dulbecco Modified Eagle’s Medium (DMEM, PAA, Austria), containing penicillin/streptomycin (100 U/ml-100 μg/ml) supplemented with 10% fetal bovine serum and kept at 37°C in an atmosphere of 5% CO2. Cells were detached from culture flasks by trypsinization and centrifuged; cell number and viability were assessed with the trypan-blue dye exclusion test.
Saos-2 were plated at a density of 2.5 × 104 cells/well in 24-well plates. 24 h after seeding, different concentrations of nanoparticles were added to the cell culture (2000 μg/ml; 1000 μg/ml; 500 μg/ml; 200 μg/ml). Nanoparticles were sonicated and vortexed before being added to the cells. Cells were incubated under standard conditions (37 °C, 5% CO2) with cell culture medium supplemented with 10 μg/ml ascorbic acid (Sigma) and 5 mM β-glycerophosphate (Sigma) for osteoblast activation, for 1, 3 and 7 days. Culture media was changed every other day. The experiments were conducted either with or without applying a static magnetic field (SMF) of 320 mT (MagnetoFACTOR-24, Chemicell, Germany) under the plates. All cell handling procedures were performed in a sterile laminar flow hood.
Live/Dead Viability/Cytotoxicity assay kit for mammalian cells (Invitrogen) was performed according to manufacturer's instructions. Briefly, cells were washed with 1x PBS for 5 min and incubated with Calcein acetoxymethyl (Calcein AM) 2 μM plus Ethidium homodimer-1 (EthD-1) 4 μM for 15 min at 37°C in the dark. Cells were washed with 1x PBS for 5 min and images acquired by a confocal microscope Fluoview FV1000 (Olympus). The live and dead cells ratio was determined by quantifying the number of cells in 3 fields at the same magnification for each HA and FeHA nanoparticles concentration at each time point without magnetic field application. Two samples per time point were analysed for each group.
Cell proliferation assay
Total DNA content was quantified using the Quanti-iTTM PicoGreen dsDNA Assay Kit (Invitrogen) assay following the manufacturer’s suggested protocol. Seeded wells were washed with 1x PBS and then incubated with 1 ml 1x PBS with 0.1% (v/v) Triton-X for cell lysis. Collected cells were centrifuged at 11000 rpm for 1 min. 25 μL of supernatant was added to 175 μL of PicoGreen® reagent working solution in a 96-well plate. Fluorescence of the samples was measured with a microplate reader (Tecan, Research Triangle Park, NC) with excitation and emission wavelengths of 485 and 535 nm, respectively. A standard curve of fluorescence versus DNA concentration was created, from which the DNA concentration values for each sample were determined. The total number of cells in the sample was determined by converting the total DNA concentration to cell number using the conversion factor of 7.7 pg DNA/cell . Five samples per condition per time point were analysed.
Alkaline phosphatase assay
Cell Alkaline Phosphatase (AP) activity was quantified using an enzymatic assay based on the hydrolysis of p-nitrophenyl phosphate (pNP-PO4) to p-nitrophenol (pNP) . Briefly, 25 μl of cell lysate, obtained as previously described (see the “cell proliferation assay” paragraph), was added to pNP-PO4 solution (Sigma-Aldrich) and allowed to react at 37°C. Absorbance was read at 0, 1, 2 and 3 min at λmax of 405 nm, using a microplate reader (Tecan, Research Triangle Park, NC) and AP activity calculated by cross-reference to a standard curve of nanomoles of p-nitrophenol liberated per cell. AP activity was normalized to total cellular content, as measured by the Picogreen assay. Five samples were analysed per condition per each time point.
In vivo pilot experiment and histological analysis
The study was performed in accordance with EC guidelines (EC Council Directive 86/609, 1986) and the Italian legislation on animal experimentation (Decreto L. vo 116/92). The research protocol on animals has been approved by the Ethical Committee of Rizzoli Orthopaedic Institute and by the responsible public authorities. Six male rabbits (Oryctolagus cuniculus, Charles River, Lecco, Italy), 2.4 ± 0.2 kg body weight, were housed at a controlled temperature of 22 ± 1°C and relative humidity of 55 ± 5% in single boxes and fed a standard diet (Mucedola, Milano, Italy) with filtered tap water ad libitum. After quarantine of at least 10 days, the animals were fasted for 24 hours before surgery. The animals were subjected to surgery to implant the tested biomaterials at the distal femoral epiphysis under general anaesthesia and in aseptic conditions. After having shaved and disinfected the posterior legs, the animals underwent a lateral longitudinal incision of lateral femoral condyle. Femoral lateral condyle trabecular bone was cross-sectionally drilled at low speed and a profuse irrigation with cold sterile 0.9% NaCl solution was maintained throughout the process to prevent the risk of bone necrosis. A critical bone defect of 6.00 mm in diameter and 8.00 mm in depth was made in each lateral femoral condyle. Defects were filled with FeHA granules and with the HA granules in the contralateral condyle as a control group. For each defect, approximately 0.5 g granules, sterilized by 25 kGy γ-ray radiation, were used. Finally, the skin was sutured. General anaesthesia was induced by an intramuscular injection of 44 mg/kg ketamine (Imalgene 1000, Merial Italia S.p.A, Milan, Italy) and 3 mg/kg xylazine (Rompun, Bayer SpA, Milano, Italy) under assisted ventilation with O2/N2O (1/0.4 l/min) mixture and 2.5% isofluorane (Forane, Abbot SpA, Latina, Italy).
Post-operatively, antibiotics and analgesics were administered: 0.6 ml/kg flumequil (Flumexil, (FATRO SpA, Bologna, Italy) and 0.1 ml/kg/day metamizole sodium (Farmolisina, Ceva Vetem SpA, Monza-Brianza, Italy).
At 4 weeks after surgery, the animals were pharmacologically euthanized with intravenous administration of Tanax (Hoechst, Frankfurt am Main, Germany), under general anaesthesia. The operated bone segments were excised and stripped of soft tissue and the presence of haematomas, edema and inflammatory tissue reactions were macroscopically evaluated. The bone segments were fixed in 4% buffered paraformaldehyde for 24 hours, dehydrated in a graded series of alcohol and finally embedded in a methyl methacrylate resin (Merck Schuchardt OHG, Hohenbrunn, Germany). Using a saw microtome (Leica SP1600, Leica Microsystems Srl, Italy), three consecutive central sections to the major axis of the implant for each bone segment were cut (60 ± 20 μm) and polished (Struers Dap-7, Struers Tech A/S, Rodovre/Copenaghen, Denmark). Then, thinned sections (30 ± 10 μm) were stained with Toluidine Blue, Acid Fucsin and Fast Green.
Results were expressed as MEAN±SEM plotted on graph (n = 5). Statistical analysis was performed using two-way ANOVA, followed by Bonferroni’s post-hoc test, for the analysis of magnetic field effect, time effect and magnetic field versus time effect. Analysis of differences between groups, for each time point, was performed by one-way ANOVA, followed by Tukey’s post-hoc test. All statistical analyses made were performed using GraphPad Prism software (version 5.0), with α=0.05.