Materials

Phosphoric acid (H3PO4, 85% pure), calcium hydroxide (Ca(OH)2, 95% pure), magnesium chloride (MgCl2∙6H2O, 85% pure), medium molecular weight chitosan (CH) in powder form ([η] = 815 mL/g, which corresponds to a viscosimetric molecular weight equal to 260 000, deacetylation degree > 75%), paraformaldehyde (PFA), Triton X-100, and magnesium chloride were purchased from Merck KGaA (Darmstadt, Germany). Ethanol was from Carlo Erba, Italy. All reagents and chemicals were of high purity grade. Ultrapure water (0.22 mS, 25 °C) was used in all experiments.

Biomineralization of chitosan with ions-doped hydroxyapatite nanoparticles and preparation of hydroxyapatite nanoparticles

To devise biocompatible, biomimetic, and bioactive particles for efficient bone regeneration, two main bio-inspired components, i.e. HA nanoparticles and chitosan, were selected and combined through a wet-chemical synthetic approach. These two components were combined according to a biomineralization approach – a process according to which it is possible to produce HA nanoparticles directly on polymer chains – and two bioactive ions, i.e. magnesium and iron, were introduced in a wet acid-base neutralization chemical route starting from calcium, magnesium, iron, and phosphate ions (Scheme 1). Specifically, an acidic solution of chitosan was dropped into a basic suspension containing calcium and the appropriate ions (magnesium or iron ions) promoting the formation of doped HA nanoparticles directly on chitosan molecules, and which are named MgHA/CH in the case of magnesium doping, whereas FeHA/CH in the case of iron doping. The same protocol was adjusted to synthesize naked – i.e. without chitosan – HA nanoparticles doped with magnesium (MgHA) or iron (FeHA) and used as a reference for successive characterization. The synthesis conditions - temperature, the ratio between the ions and the rate of addition of the reagents - were set to obtain nanometric particles with low crystallinity, whose visual aspect in the form of powder is reported in Fig. 1A, whereas in the form of nanoparticles suspension is reported in Fig. 1B, confirming the synthesis of aqueous dispersions with different colors, i.e. white in the case of magnesium doping, whereas brown in the case of brown doping, in mineralized and naked particles.

Scheme 1

The schematic synthesis of HA-based nanoparticles and the biomineralization process according to which it is possible to synthesize HA-based nanoparticles on chitosan chains. Specifically, in (A), the wet acid-base neutralization of acidic chitosan solution containing phosphate ions in a basic suspension containing calcium ions – and additional doping ions, i.e. Mg2+, Fe2+, and Fe3+ – is reported, leading to the biomineralization of chitosan (B). In (C) the magnification of the schematic formation of hydroxyapatite nanoparticles doped with ions on the chitosan template is reported. Created with Biorender.com

In detail, Mg-doped hydroxyapatite nanoparticles (MgHA) were prepared at environmental temperature by wisely dropping in 90 min 150 mL of an aqueous solution containing 20.63 g of H3PO4 (Merck KGaA, Darmstadt, Germany, 85% pure) into a basic suspension consisting of 23.3 g Ca(OH)2 (Merck KGaA, Darmstadt, Germany, 95% pure) and 3.05 g MgCl2∙6H2O (Merck KGaA, Darmstadt, Germany, 85% pure) in 200 mL of water under stirring. The resulting suspension was maintained under stirring for two hours, then left at rest overnight.

1 g of chitosan was solubilized in 200 mL of an aqueous solution containing 1.61 g of H3PO4 overnight under stirring. Then the resulting suspension was added to a basic suspension consisting of 1.81 g Ca(OH)2 and 240 mg MgCl2∙6H2O (Merck KGaA, Darmstadt, Germany, 85% pure) in 200 mL of water under stirring. The resulting pellet consisting of chitosan mineralized with Mg-doped hydroxyapatite nanoparticles (MgHA/CH) was maintained at rest for 2 h.

Fe-doped hydroxyapatite nanoparticles (FeHA) were prepared at 40 °C by wisely dropping in 90 min 150 mL of an aqueous solution containing 20.63 g of H3PO4 (Merck KGaA, Darmstadt, Germany, 85% pure) into a basic suspension consisting of 23.3 g Ca(OH)2 (Merck KGaA, Darmstadt, Germany, 95% pure) and 6.02 g FeCl2∙6H2O (Merck KGaA, Darmstadt, Germany, 85% pure) and 8.07 g FeCl3∙6H2O (Merck KGaA, Darmstadt, Germany, 85% pure) in 200 mL of water under stirring. The resulting suspension was maintained under stirring for two hours, then left at rest overnight.

1 g of chitosan was solubilized in 200 mL of an aqueous solution containing 1.61 g of H3PO4 overnight under stirring. Two solutions of iron, i.e. 0.47 g FeCl2∙6H2O (Merck KGaA, Darmstadt, Germany, 85% pure) and 0.63 FeCl3∙6H2O (Merck KGaA, Darmstadt, Germany, 85% pure), both of them separately solubilized in 20 mL of water were rapidly added to a basic suspension under stirring at 40 °C consisting of 1.81 g Ca(OH)2 in 200 mL of water. Then the solution of chitosan was slowly added to the basic suspension under stirring at 40 °C and was maintained under stirring for 2 h at the same temperature. The resulting suspension consisting of chitosan mineralized with Fe-doped hydroxyapatite nanoparticles (FeHA/CH) was maintained at rest for 2 h.

Finally, all the resulting suspensions were precipitated by centrifugation and successive resuspension (by sonication) and washed three times. All the resulting slurries were frozen, freeze-dried, and finally sieved with 200 μm mesh before successive characterization.

Inductively Coupled Plasma-Optical Emission Spectrometry

Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES, Agilent Technologies 5100 ICP-OES, Santa Clara, USA) was used for the quantitative determination of Mg2+, Ca2+, and PO43− ions that constituted the inorganic mineral component. Briefly, 20 mg of each sample were dissolved in 2 mL nitric acid (65 wt%) followed by subsequent sonication and dilution with 100 mL of distilled water.

FTIR-ATR spectroscopy

Fourier transform infrared (FT-IR) spectra of the samples were measured with a Nicolet iS™ 50 FT-IR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) using the Attenuated Total Reflection (ATR) mode. Each IR spectrum was acquired using a 2 cm− 1 resolution in a spectral window ranging from 500 to 4000 cm− 1 with 100 scans.

X-ray diffraction

X-ray diffraction (XRD) patterns were recorded by a Bruker (Karlsruhe, Germany) AXS D8 Advance diffractometer in reflection mode, with CuK radiation (\(\:\lambda\:\) = 1.54178 Å) generated at 40 kV and 40 mA and equipped with a Lynx-eye position-sensitive detector. XRD spectra were recorded in the range from 20° to 60° with a step size (2θ) of 0.02° and a counting time of 0.5 s.

Scanning Electron Microscopy (SEM)

Nanoparticles morphologies were investigated by a Zeiss EVO40 Scanning Electron Microscope (SEM) equipped with EDX (Energy-Dispersive X-ray analyses). Samples were mounted on aluminum stubs covered by adhesive carbon tape and gold-sputtered before SEM analyses at 20.0 kV.

Thermogravimetric analyses

Thermogravimetric analyses (TGA) were performed using an STA 449/C Jupiter (Netzsch, Germany) on 10 mg of sample, placed in an alumina crucible under airflow, and brought from room temperature to 1100 °C at a heating rate of 10 °C/minute.

In vitro biological tests

Two different bone cell models were used for in vitro biological tests including human osteoblasts-like cells HOBIT (Human OsteoBlast-like Initial Transfectant, RRID: CVCL_W632) and human osteosarcoma osteoblast-like cells MG63 (ATCC CRL-1427™). HOBIT and MG63 cells were grown using Advanced DMEM/F12 (Gibco, Thermo Fisher Scientific, USA) both supplemented with 10% v/v Fetal Bovine Serum (FBS) and 1% v/v penicillin/streptomycin (100 U/mL-100 µg/mL) at 37 °C in an atmosphere with 5% CO2. Cells were detached by using trypsin-EDTA (0.25% (w/v) trypsin and 0.03% EDTA) and centrifuged, and the number of viable cells was assessed with a trypan blue dye exclusion test in an automatic CytoSMART Corning (New York, USA) system before seeding in multi-well plates for biological experiments. Cells exposed to H2O2 1 mM in OptiMEM (Gibco, Thermo Fisher Scientific, USA) were used positive control cells.

Sterilization of nanoparticle suspension for biological tests

10 mg of ions-doped hydroxyapatite nanoparticles (MgHA and FeHA) were transferred in 1.5 mL Eppendorf tubes with 1 mL of ethanol 70% for 20 min in a sonicating bath. In the case of chitosan biomineralized with ions-doped hydroxyapatite nanoparticles the amount of powder was adjusted (considering the percentage of inorganic components determined by TGA analyses) to have the same amount of hydroxyapatite nanoparticles in all samples; specifically, for chitosan biomineralized with Mg-doped hydroxyapatite nanoparticles, MgHA/CH, 12.8 mg, whereas for chitosan biomineralized with Fe-doped hydroxyapatite nanoparticles, FeHA/CH, 14.9 mg were used. Thereafter, Eppendorf tubes were centrifugated at 12 000 g for 5 min with EBA 12R (Hettich GmbH & Co. KG, Tuttlingen, Germany) to promote the formation of a pellet containing nanoparticles, the supernatant was discarded, 1 mL of sterilized deionized water was added, and nanoparticles were resuspended by using a sonicating bath for 15 min. Finally, nanoparticles were diluted in the appropriate cell culture media to achieve final hydroxyapatite concentrations equal to 200 µg/mL. The composition of cell media was adjusted to have the 10% V/V of deionized water in all samples, including the control.

Cell viability assay

The cellular viability and proliferation of cells were assessed by the PrestoBlue™ Cell Viability Reagent procedure (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, cells were seeded (2.0 × 103 cells/well) in a 96 plate and the next day supernatant was substituted with 100 µL of HA-based nanoparticles diluted in cell culture media. Untreated cells were used as a negative control. Two time points (1 day, 3 days) were evaluated. At the desired timeframe, 10% PrestoBlue Reagent was added for two hours at 37 °C and 5% CO2 atmosphere in dark conditions under controlled humidity conditions. After incubation, the samples were read at excitation and emission wavelengths of 544 and 590 nm, respectively, by using the BioTek Synergy H1 Multimode Reader (Agilent, USA). At least three replicates for each sample were performed.

Cell counting assay

50,000 cells were seeded in 6-well plates and after letting them overnight at rest, 2 mL of HA-based nanoparticles diluted in cell culture media were added. At the desired timeframe, i.e. after 1, 3, and 7 days of incubation with particles, cells were washed in PBS 1×, detached by using trypsin, and the cell viability were assessed by cell counting with a trypan blue dye exclusion test in an automatic CytoSMART Corning (New York, USA) system.

Clonogenic assay

For clonogenic assay, 1 million cells were seeded in 6-well plates and after 1 day media was replaced with 2 mL of HA-based nanoparticles diluted in cell culture media. After 1 day of incubation with nanoparticles, cells were washed in PBS 1×, detached by using trypsin, and 750 cells were seeded in 6-well plates. Cells were allowed to proliferate and form colonies for 7 days – replacing the media with fresh media every 2–3 days – after which colonies were washed in PBS 1×. Thereafter, colonies were stained by using 800 µL of 10% w/V crystal violet (Merck KGaA, Darmstadt, Germany) in 70% V/V ethanol, washed three times by using deionized water, and let dry. Finally, the number of colonies in each plate was determined by using the ImageQuant TL (Amersham) scanner and its software.

Reactive oxygen species determination

The production of reactive oxygen species by cells was assessed in 96 well-plates (2.0 × 103 cells/well) according to a previously reported protocol with slight modifications [33, 34]. After overnight incubation, the supernatant was discarded and replaced with the nanoparticles diluted in the cell culture media. Cells without nanoparticles were used as a negative control. Reduced glutathione (GSH, Merck KGaA, Darmstadt, Germany) treatment (10 mM), and GSH combined with FeHA/CH, were performed for 1 day as control samples. After 1 day of incubation, supernatants were discarded and cells were washed in PBS 1×, then cells were incubated with 100 µL of CM-H2DCFDA (ThermoFisher Scientific, USA) 5 µM diluted in OptiMEM medium. Cells were incubated for 30 min in dark conditions, then supernatants were discarded, and cells were washed in PBS 1× and incubated for 30 min in 100 µL of OptiMEM in dark conditions. Finally, multi-well plates were read at excitation and emission wavelengths of 494 and 522 nm, respectively, by using BioTek SynergyH1 (Agilent, USA). At least three replicates for each condition were performed.

Immunofluorescence

80 000 MG63 cells were seeded in 24-well plates and after overnight incubation media was replaced with 500 µL of HA-based nanoparticles diluted in cell culture media. After 1 day of incubation with nanoparticles, cells were washed in PBS 1× and fixed in 4% paraformaldehyde. Cells were permeabilized with 0.25% Triton X-100 in PBS 1× for 5 min. After blocking overnight with 10% FBS in Washing Buffer (Tris HCl 10 mM, pH 7.4, NaCl 150 mM, and Tween 20 0.01%), cells were incubated with the primary antibody anti-γH2A.X (monoclonal antibody, IgG mouse, #05-636 Millipore, Merck, Darmstadt, Germany) diluted 1:500 V/V in blocking solution (10% FBS in Washing Buffer) for 2 h at 37 °C.

Then, cells were incubated with goat Anti-Mouse Alexa Fluor® 555-conjugated secondary antibodies (Abcam, Cambridge, UK) for 1 h at 37 °C. Cells were incubated with DAPI (Thermo Fisher Scientific, USA) 14.3 mM for 5 min at room temperature for nuclear staining. Cells were finally washed and mounted with Mowiol mounting medium with antifading agent 1,4-Diazobicyclo- [2]-octane (DABCO™). Cells were visualized through a Leica TCS SP8 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a stage-top environmental chamber (Okolab, Italy) and operated by Leica Application Suite X (LAS X) 3.5.5 software. Images were collected as z-stacks using a 100 x/1.4 oil immersion objective, a 405 nm diode laser (DAPI excitation), and a tunable white-light laser (λex: 555 nm) and are reported as maximum intensity projections. Determination of the fluorescent signal was performed using the FIJI IMAGEJ Software (National Institute of Health, Bethesda, MD, USA).

Western blot analyses

For western blotting analyses, 1 million cells were seeded in 6-well plates. After overnight incubation, media was replaced with 2 mL of HA-based nanoparticles diluted in cell culture media. After 1 day of incubation with nanoparticles, cells were washed in PBS 1×, detached by using trypsin, recovered by centrifugation and the pellet was transferred in a 1.5 mL Eppendorf tube and lysed by using a solution containing 0.1% Triton X-100 and protease inhibitors (dithiothreitol (DTT) 1 mM, phenylmethylsulfonyl fluoride (PMSF) 2.5 mM, protease inhibitor cocktail (PI) 0.001% V/V, NaF 1 mM, and Na3VO4 10 mM) in PBS 1× for 10 min in an ice bath, centrifugated at 12 000 g for 30 min at 4 °C with EBA 12R (Hettich GmbH & Co. KG, Tuttlingen, Germany) and stored at -80 °C. The concentration of proteins in the cell lysates (supernatant) was determined by colorimetric Bradford assays (Bio-Rad, Hercules, USA), and 30 µg of proteins were diluted in Laemmli buffer (final loaded volume for each well equal to 20 µL), denatured at 95 °C for 5 min, resolved on 15% SDS/PAGE, transferred onto nitrocellulose membranes (Schleicher & Schuell Bioscience, Dassel, Germany), and incubated overnight with β-tubulin (monoclonal antibody, IgG1 mouse, Merck, Darmstadt, Germany) 1:2000, PRXSO3 1:1000 (polyclonal antibody, IgG rabbit, Abcam, Cambridge, UK) and γH2AX 1:1000 (monoclonal antibody, IgG mouse, #05-636 Millipore, Merck, Darmstadt, Germany) antibodies. The corresponding secondary antibodies labeled with IR-Dye (goat anti-rabbit IgG IRDye 680 for PRXSO3, goat anti-mouse IgG IRDye 800 for γH2AX, and goat anti-mouse IgG IRDye 680 for β-tubulin) were used (1:10 000) and incubated for 1 h. PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa, was used to assess the molecular weight of the corresponding protein. Images were acquired and quantified by using an Odyssey CLx Infrared Imaging System (LI-COR Biosciences). Cells incubated with H2O2 1 mM for 10 min in OptiMEM (Gibco, Thermo Fisher Scientific, USA) were used as positive control cells for γH2AX analyses, whereas cells incubated with H2O2 1 mM for different timeframes including 2, 4, 8, and 10 min in OptiMEM (Gibco, Thermo Fisher Scientific, USA) were used as positive control cells for PRXSO3 analyses.

Alkaline COMET assay

60 000 HOBIT cells were plated on 24-well plates, and after overnight incubation media was replaced with 2 mL of HA-based nanoparticles diluted in cell culture media. Reduced glutathione (GSH, Merck KGaA, Darmstadt, Germany) treatment (10 mM) was performed for 1 day as control cells. After 1 day of incubation with nanoparticles, cells were washed in PBS 1×, detached by using 50 µL of trypsin for 10 min, recovered by pipetting and adding 200 µL of complete media. The pellet was mixed with 1 mL of low melting point agarose 1% w/V (LE GQ Agarose, MBI Fermentas, USA) in PBS 1× pre-heated at 40 °C. One milliliter of the mixture was applied on a SuperFrost Menzel (Thermo Scientific, USA) glass coverslip pre-coated with a thin layer (800 µL) of normal melting point agarose 1% w/V (Merck KGaA, Darmstadt, Germany) in deionized water (let dry at 4 °C overnight). Cells were then lysed with cold alkaline lysis buffer (NaCl 2.5 M, EDTA disodium salt 100 mM, Tris base 10 mM, 1% DMSO, 1% Triton X-100; pH 10.5) for 1 h at 4 °C. Next, cells were transferred to an electrophoretic cell (Hoefer supersub, Hoefer Inc, USA) and covered with alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, 1% DMSO; pH > 13) for 30 min for the DNA to denature. DNA electrophoresis was carried out at 25 V and 300 mA for 25 min at room temperature. Coverslips were washed with neutralization buffer (Tris-HCl 500 mM, pH 8.1) for 5 min at room temperature three times and let dry at 60 °C for 2 h. DNA was then stained with 1 mL of SYBR™ Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, USA) solution 1× (diluted 1:10 000 in deionized water) for 30 min at room temperature in the dark and let dry at 60 °C for 2 h. Leica TCS SP8 laser-scanning confocal microscope (Leica Microsystems) was used for comet analysis. The olive tail moment was measured by the IMAGEJ Software (National Institute of Health, Bethesda, MD, USA) plugin OPENCOMET.

Statistical analysis

Statistical analysis and graph elaboration were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA). One-way ANOVA (analysis of variance) was performed followed by Dunnett post hoc tests to evaluate differences among different groups and control or Tuckey post hoc tests to evaluate differences among all groups. Differences were considered significant for p-values less than 0.05.